Anti-tnf-alpha-antibodies and functional fragments thereof

ABSTRACT

The present invention relates to antibody molecules and functional fragments thereof, capable of binding to tumor necrosis factor alpha (TNFα), to processes for their production, and to their therapeutic uses.

FIELD OF THE INVENTION

The present invention relates to antibody molecules and functionalfragments thereof, capable of binding to tumor necrosis factor alpha(TNFα), to processes for their production, and to their therapeuticuses.

BACKGROUND

TNFα is a homo-trimeric pro-inflammatory cytokine that is released byand interacts with cells of the immune system. TNFα has also been shownto be up-regulated in a number of human diseases, including chronicdiseases such as rheumatoid arthritis, Crohn's disease, ulcerativecolitis and multiple sclerosis.

Antibodies to TNFα have been proposed for the prophylaxis and treatmentof endotoxic shock (Beutler et al., Science, 234, 470-474, 1985). Bodmeret al., (Critical Care Medicine, 21, S441-S446, 1993) and Wherry et al.,(Critical Care Medicine, 21, S436-S440, 1993) discuss the therapeuticpotential of anti-TNFα antibodies in the treatment of septic shock. Theuse of anti-TNFα antibodies in the treatment of septic shock is alsodiscussed by Kirschenbaum et al., (Critical Care Medicine, 26,1625-1626, 1998). Collagen-induced arthritis can be treated effectivelyusing an anti-TNFα monoclonal antibody (Williams et al. (PNAS-USA, 89,9784-9788, 1992)).

The use of anti-TNFα antibodies in the treatment of rheumatoid arthritisand Crohn's disease is discussed in Feldman et al. (TransplantationProceedings, 30, 4126-4127, 1998), Adorini et al. (Trends in ImmunologyToday, 18, 209-211, 1997) and in Feldman et al. (Advances in Immunology,64, 283-350, 1997). The antibodies to TNFα previously used in suchtreatments are generally chimeric antibodies, such as those described inU.S. Pat. No. 5,919,452.

Monoclonal antibodies against TNFα have been described in the prior art.Meager et al. (Hybridoma, 6, 305-311, 1987) describe murine monoclonalantibodies against recombinant TNFα. Fendly et al. (Hybridoma, 6,359-370, 1987) describe the use of murine monoclonal antibodies againstrecombinant TNFα in defining neutralising epitopes on TNFα.

Furthermore, in International Patent Application WO 92/11383,recombinant antibodies, including CDR-grafted antibodies, specific forTNFα are disclosed. Rankin et al. (British J. Rheumatology, 34, 334-342,1995) describe the use of such CDR-grafted antibodies in the treatmentof rheumatoid arthritis. U.S. Pat. No. 5,919,452 discloses anti-TNFαchimeric antibodies and their use in treating pathologies associatedwith the presence of TNFα. Further anti-TNFα antibodies are disclosed inStephens et al. (Immunology, 85, 668-674, 1995), GB-A-2 246 570, GB-A-2297 145, U.S. Pat. No. 8,673,310, US 2014/0193400, EP 2 390 267 B1, U.S.Pat. Nos. 8,293,235, 8,697,074, WO 2009/155723 A2 and WO 2006/131013 A2.

The prior art recombinant anti-TNFα antibody molecules generally have areduced affinity for TNFα compared to the antibodies from which thehypervariable regions or CDRs are derived. All currently marketedinhibitors of TNFα are administered intravenously or subcutaneously inweekly or longer intervals as bolus injections, resulting in highstarting concentrations that are steadily decreasing until the nextinjection.

Currently approved anti-TNFα biotherapeutics include (i) infliximab, achimeric IgG anti-human monoclonal antibody (Remicade®; Wiekowski M etal: “Infliximab (Remicade)”, Handbook of Therapeutic Antibodies,WILEY-VCH; Weinheim, 2007-01-01, p. 885-904); (ii) etanercept, a TNFR2dimeric fusion protein, with an IgG1 Fc (Enbrel®); (iii) adalimumab, afully human monoclonal antibody (mAb) (Humira®; Kupper H et al:“Adalimumab (Humira)”, Handbook of Therapeutic Antibodies, WILEY-VCH;Weinheim, 2007-01-01, p. 697-732); (iv) certolizumab, a PEGylated Fabfragment (Cimzia®; Melmed G Y et al: “Certolizumab pegol”, NatureReviews. Drug Discovery, Nature Publishing Group, GB, Vol. 7, No. 8,2008-08-01, p. 641-642); (v) Golimumab, a human IgGIK monoclonalantibody (Simponi®; Mazumdar S et al: “Golimumab”, mAbs, LandesBioscience, US, Vol. 1, No. 5, 2009-09-01, p. 422-431). However, variousbiosimilars are in development, and a mimic of infliximab known asRemsima has already been approved in Europe.

Infliximab has a relatively low affinity to TNFα (K_(D)>0.2 nM; Weir etal., 2006, Therapy 3: 535) and a limited neutralization potency in anL929 assay. In addition, infliximab shows substantially nocross-reactivity with TNFα from Cynomolgus or Rhesus monkeys. Foranti-TNFα antibodies, however, cross-reactivity with TNFα from monkeysis highly desirable, as this allows for animal tests with primates,reflecting the situation in humans in many aspects.

Etanercept, although a bivalent molecule, binds TNFα at a ratio of onetrimer per one etanercept molecule, precluding the formation of largeantigen-biotherapeutics complexes (Wallis, 2008, Lancet Infect Dis, 8:601). It does not inhibit LPS-induced cytokine secretion in monocytes(Kirchner et al., 2004, Cytokine, 28: 67).

The potency of adalimumab is similar to that of infliximab. Anotherdisadvantage of adalimumab is its poor stability, e.g. as determined ina thermal unfolding test. The melting temperature (T_(m)) of adalimumabin such a test was determined to be 67.5° C. The lower the T_(m) valueof an antibody, however, the lower is its general stability. A lowerT_(m) makes antibodies less suitable for pharmaceutical use, e.g. fororal administration.

The potency of certolizumab is slightly greater than that of infliximab,but still not satisfying. Certolizumab does not inhibit T-cellproliferation in a MLR (Vos et al., 2011, Gastroenterology, 140: 221).

EP2623515 A1 discloses humanized anti-TNFα antibodies andantigen-binding fragments (Fab) thereof. As becomes clear from thedisclosed examples, the potency of the resulting humanized Fab fragmentsis comparable to that of infliximab in a L929 neutralization assay (seeTable 2 and 5). The sole anti-TNFα IgG antibody tested forcross-reactivity binds only weakly to Rhesus TNF-α (see [0069]; FIG. 3).Cross-reactivity with Cynomolgus TNFα was not tested. Moreover, there isweak binding to human TNFβ (see FIG. 3). Therefore, EP2623515 A1 doesnot disclose anti-TNFα antibodies or functional fragments thereof, whichhave a potency to inhibit TNFα-induced apoptosis in L929 cells greaterthan that of infliximab and which are cross-reactive with Rhesus TNFαand Cynomolgus TNFα.

WO 2012/007880 A2 discloses a modified single domain antigen bindingmolecule (SDAB) in the form of fusion proteins comprising one or moresingle antigen binding domains that bind to one or more targets (e.g.TNFα), a linker and one or more polymer molecules. The only specificexample given is termed SDAB-01 and includes two antigen bindingdomains, which bind to TNFα, connected with a flexible linker, and aC-terminal Cysteine supporting the site specific PEGylation (see FIG.3). WO 2012/007880 A2 fails to compare the potency of SDAB-01 to knownTNFα antibodies like infliximab in a L929 cell-based neutralizationassay, or to assess other SDAB-01-specific parameters like theeffectiveness to block TNFα-TNFRI/II interaction and the selectivity forbinding TNFα over TNFβ. In an assay where the treatment with SDAB-01 andinfliximab are compared in a transgenic mouse model for polyarthritisthat overexpresses human TNFα (see page 54, Example 8), the two seem tobe similarly effective in preventing further development of arthritis(e.g. FIGS. 17 & 18). However, the dosage given in this example ismisleading as the molecular weight of SDAB-01 is less than half of thatof infliximab. Thus, WO 2012/007880 A2 does not disclose anti-TNFαantibodies having a potency to inhibit TNFα-induced apoptosis in L929cells greater than that of infliximab.

WO 2015/144852 A1 investigates the properties of an anti-TNF-α scFvdesignated “scFv1”. This scFv showed a TNFα neutralization capacity in aPK-15 cell assay that was comparable to that of infliximab (see [0236]).In addition, the scFv seems to have some cross-reactivity to TNF-α fromrhesus macaque and cynomolgus monkey (see Ex. 8). No affinity data arereported in WO 2015/144852 A1. The single chain antibody fragment DLX105(also known as ESBA 105), however, which is known to have only moderateaffinity (K_(D)=157 pM; see Urech et al. 2010 Ann Rheum Dis 69: 443),shows a better binding to TNF-α than scFv1 (see FIG. 1 of WO 2015/144852A1). Therefore, WO 2015/144852 A1 does not disclose anti-TNF-αantibodies having high affinity for human TNFα (K_(D)<125 pM).

WO 2015/065987 A1 describes anti-TNF-α antibodies, anti-IL-6 antibodies,and bispecific antibodies binding to both antigens. Certain anti-TNFαantibodies showed some cross-reactivity with TNFα from Cynomolgus (FIG.17). The anti-TNFα antibodies, however, exhibited a significantly lowerpotency than infliximab in an L929 neutralization assay ([0152]; FIG.5). Therefore, WO 2015/065987 A1 does not disclose anti-TNF-α antibodieshaving a potency to inhibit TNFα-induced apoptosis in L929 cells greaterthan that of infliximab.

Drugs in R&D, Vol. 4 No. 3, 2003, pages 174-178 desribes the humanizedantibody “Humicade” (CDP 571; BAY 103356), a monoclonal anti-TNFαantibody with high affinity. The potency of Humicade to inhibitTNFα-induced apoptosis in L929 cells, however, appears to be limited(see, e.g., US 2003/0199679 A1 at [0189]). The reference therefore doesnot disclose anti-TNF-α antibodies having a potency to inhibitTNFα-induced apoptosis in L929 cells greater than that of infliximab.

Saldanha J W et al: “Molecular Engineering I: Humanization”, Handbook ofTherapeutic Antibodies, Chapter 6, 2007-01-01, WILEY-VCH, Weinheim, p.119-144 discloses different strategies for humanization of monoclonalantibodies including CDR Grafting, Resurfacing/Veneering, SDR transferand Delmmunization Technology.

There is a need for improved antibody molecules to treat chronicinflammatory diseases such as inflammatory bowel disorders. The antibodymolecules should at least have (i) high affinity for human TNFα (i.e. aK_(D)<125 pM), (ii) a high potency to inhibit TNFα-induced apoptosis inL929 cells, (iii) a high potency to inhibit LPS-induced cytokinesecretion, (iv) substantial affinity to TNFα from Cynomolgus and Rhesus(e.g. a K_(D)<1 nM), and (v) a high melting temperature of the variabledomain as determined in a thermal unfolding experiment (e.g. a T_(m)>70°C.).

SUMMARY OF THE INVENTION

The inventors of the present application found that certain anti-TNFαantibodies and functional fragments thereof exhibit a combination offavorable properties, including high affinity for human TNFα (K_(D)<125pM), a potency to inhibit TNFα-induced apoptosis in L929 cells greaterthan that of infliximab, a potency to inhibit LPS-induced cytokinesecretion greater than that of adalimumab, and substantial affinity(K_(D)<1 nM) to TNFα from animals such as Cynomolgus monkey (Macacafascicularis) and/or Rhesus macaques (Macaca mulatta). In addition, theantibodies and functional fragments thereof were specific for TNFα inthat they did not significantly bind to TNFβ, and exhibit a significantstability, as determined in a thermal unfolding assay of the variabledomain.

The invention provides antibody molecules and functional fragmentsthereof.

The present invention therefore relates to the subject matter defined inthe following items (1) to (48):

-   (1) An antibody or a functional fragment thereof capable of binding    to human tumor necrosis factor alpha (TNFα), wherein said antibody    or functional fragment thereof comprises (i) a V_(L) domain    comprising a CDR1 region having an amino acid sequence in accordance    with the amino acid sequence as shown in SEQ ID NO:1, a CDR2 region    having an amino acid sequence in accordance with the amino acid    sequence as shown in SEQ ID NO:2, and a CDR3 region having an amino    acid sequence in accordance with the amino acid sequence as shown in    SEQ ID NO:3, and (ii) a V_(H) domain comprising a CDR1 region having    an amino acid sequence in accordance with the amino acid sequence as    shown in SEQ ID NO:4, a CDR2 region having an amino acid sequence in    accordance with the amino acid sequence as shown in SEQ ID NO:5, and    a CDR3 region having an amino acid sequence in accordance with the    amino acid sequence as shown in SEQ ID NO:6.-   (2) The antibody or functional fragment of item (1), wherein said    antibody or functional fragment comprises (i) a V_(L) domain    comprising a CDR1 region having the amino acid sequence as shown in    SEQ ID NO:7, a CDR2 region having the amino acid sequence as shown    in SEQ ID NO:8, and a CDR3 region having the amino acid sequence as    shown in SEQ ID NO:9, and (ii) a V_(H) domain comprising a CDR1    region having the amino acid sequence as shown in SEQ ID NO:10, a    CDR2 region having the amino acid sequence as shown in SEQ ID NO:11,    and a CDR3 region having the amino acid sequence as shown in SEQ ID    NO:12.-   (3) The antibody or functional fragment of any one of the preceding    items, wherein said antibody or functional fragment comprises a    V_(H) domain having the amino acid sequence as shown in SEQ ID    NO:13.-   (4) The antibody or functional fragment of any one of the preceding    items, wherein said antibody or functional fragment comprises a    V_(L) domain having an amino acid sequence selected from SEQ ID    NO:14 and SEQ ID NO:54, preferably having the amino acid sequence as    shown in SEQ ID NO:14.-   (5) The antibody or functional fragment of any one of the preceding    items, wherein said antibody or functional fragment thereof    specifically binds to human TNFα.-   (6) The antibody or functional fragment of any one of any one of the    preceding items, wherein said antibody or functional fragment    thereof does not significantly bind to TNFβ.-   (7) The antibody or functional fragment of any one of the preceding    items, wherein said antibody or functional fragment    -   (i) binds to human TNFα with a dissociation constant (K_(D)) of        less than 125 pM;    -   (ii) is cross-reactive with Macaca mulatta TNFα and with Macaca        fascicularis TNFα;    -   (iii) has a greater potency than infliximab, as determined by an        L929 assay; and/or    -   (iv) is capable of binding to human TNFα_(Trimer) in a        stoichiometry (antibody: TNFα_(Trimer)) of at least 2.-   (8) The antibody or functional fragment of any one of the preceding    items, which binds to human TNFα with a K_(D) of less than 100 pM,    preferably of less than 50 pM.-   (9) The antibody or functional fragment of any one of the preceding    items, which binds to TNFα from Macaca mulatta with a K_(D) of less    than 1 nM.-   (10) The antibody or functional fragment of any one of the preceding    items, which binds to TNFα from Macaca fascicularis with a K_(D) of    less than 1 nM.-   (11) The antibody or functional fragment of any one of the preceding    items, wherein the potency of the antibody or functional fragment to    inhibit TNFα-induced apoptosis relative to that of infliximab    (relative potency), determined in an L929 assay, is greater than 5,    and wherein said relative potency is the ratio of the 10₅₀ value in    ng/mL of infliximab in the L929 assay to the 10₅₀ value in ng/mL of    the antibody in scFv format in the L929 assay.-   (12) The antibody or functional fragment of any one of the preceding    items, wherein the melting temperature of the variable domain of the    antibody in scFv format, determined by differential scanning    fluorimetry, is at least 65° C.-   (13) The antibody or functional fragment of any one of the preceding    items, wherein the melting temperature of the variable domain of the    antibody in scFv format, determined by differential scanning    fluorimetry, is at least 68° C.-   (14) The antibody or functional fragment of any one of the preceding    items, wherein the melting temperature, determined by differential    scanning fluorimetry, is at least 70° C.-   (15) The antibody or functional fragment of any one of the preceding    items, wherein the loss in monomer content, after five consecutive    freeze-thaw cycles, is less than 0.2%.-   (16) The antibody or functional fragment of any one of the preceding    items, wherein the loss in monomer content, after storage for four    weeks at 4° C., is less than 1%.-   (17) The antibody or functional fragment of any one of the preceding    items, wherein the potency of the antibody or functional fragment to    block the interaction between human TNFα and TNF receptor I (TNFRI),    relative to that of infliximab (relative potency), as determined in    an inhibition ELISA, is at least 2, wherein said relative potency is    the ratio of the IC₅₀ value in ng/mL of infliximab to the IC₅₀ value    in ng/mL of the antibody in scFv format.-   (18) The antibody or functional fragment of any one of the preceding    items, wherein the potency of the antibody or functional fragment to    block the interaction between human TNFα and TNF receptor II    (TNFRII), relative to that of infliximab (relative potency), as    determined in an inhibition ELISA, is at least 2, wherein said    relative potency is the ratio of the IC₅₀ value in ng/mL of    infliximab to the IC₅₀ value in ng/mL of the antibody in scFv    format.-   (19) The antibody or functional fragment of any one of the preceding    items, which is capable of inhibiting cell proliferation of    peripheral blood mononuclear cells in a mixed lymphocyte reaction.-   (20) The antibody or functional fragment of any one of the preceding    items, which is capable of inhibiting LPS-induced secretion of    interleukin-1β from CD14⁺ monocytes.-   (21) The antibody or functional fragment of item (20), wherein the    IC₅₀ value for inhibiting LPS-induced secretion of interleukin-1β is    less than 1 nM.-   (22) The antibody or functional fragment of item (21), wherein said    IC₅₀ value for inhibiting LPS-induced secretion of interleukin-1β,    on a molar basis, is lower than that of adalimumab.-   (23) The antibody or functional fragment of any one of the preceding    items, which is capable of inhibiting LPS-induced secretion of TNFα    from CD14⁺ monocytes.-   (24) The antibody or functional fragment of item (23), wherein the    IC₅₀ value for inhibiting LPS-induced secretion of TNFα is less than    1 nM.-   (25) The antibody or functional fragment of item (24), wherein said    IC₅₀ value for inhibiting LPS-induced secretion of TNFα, on a molar    basis, is lower than that of adalimumab.-   (26) The antibody of any one of the preceding items, which is an    immunoglobulin G (IgG).-   (27) The functional fragment of any one of items (1) to (25), which    is a single-chain variable fragment (scFv).-   (28) The functional fragment of item (27), wherein said scFv    comprises or consists of an amino acid sequence selected from SEQ ID    NO:15 and SEQ ID NO:55, preferably the amino acid sequence as shown    in SEQ ID NO:15.-   (29) The functional fragment of any one of items (1) to (25), which    is a diabody.-   (30) The functional fragment of item (29), wherein said diabody    comprises or consists of the amino acid sequence as shown in SEQ ID    NO:51.-   (31) An antibody or functional fragment thereof binding to    essentially the same epitope on human TNFα as an antibody comprising    a V_(H) domain having the amino acid sequence as shown in SEQ ID    NO:13 and a V_(L) domain having the amino acid sequence as shown in    SEQ ID NO:14, in particular wherein said antibody or functional    fragment exhibits one or more of the features referred to in    items (1) to (30) herein above.-   (32) The antibody or functional fragment of any one of the preceding    items, wherein the sum of (i) the number of amino acids in framework    regions I to III of the variable light domain of said antibody or    functional fragment that are different from the respective human Vκ1    consensus sequences with SEQ ID NOs: 56 to 58 (see Table 15),    and (ii) the number of amino acids in framework region IV of the    variable light domain of said antibody or functional fragment that    are different from the most similar human A germline-based sequence    selected from SEQ ID NOs: 59 to 62 (see Table 16), is less than 7,    preferably less than 4.-   (33) The antibody or functional fragment of any one of the preceding    items, wherein the framework regions I to III of the variable light    domain of said antibody or functional fragment consist of human Vκ1    consensus sequences with SEQ ID NOs:56 to 58, respectively, and    framework region IV consists of a λ germline-based sequence selected    from SEQ ID NOs:59 to 62.-   (34) A nucleic acid encoding the antibody or functional fragment of    any one of the preceding items.-   (35) A vector or plasmid comprising the nucleic acid of item (34).-   (36) A cell comprising the nucleic acid of item (35) or the vector    or plasmid of item (34).-   (37) A method of preparing the antibody or functional fragment of    any one of items (1) to (33), comprising culturing the cell of    item (36) in a medium under conditions that allow expression of the    nucleic acid encoding the antibody or functional fragment, and    recovering the antibody or functional fragment from the cells or    from the medium.-   (38) A pharmaceutical composition comprising the antibody or    functional fragment of any one of items (1) to (33), and optionally    a pharmaceutically acceptable carrier and/or excipient.-   (39) The antibody or functional fragment as defined in any one of    items (1) to (33) for use in a method of treating an inflammatory    disorder or a TNFα-related disorder.-   (40) The antibody or functional fragment for use according to item    (39), wherein said inflammatory disorder is selected from the list    of diseases and disorders listed in Section “Disorders to be    treated” below.-   (41) The antibody or functional fragment for use according to item    (39), wherein said inflammatory disorder is an inflammatory disorder    of the gastrointestinal tract.-   (42) The antibody or functional fragment for use according to item    (41), wherein said inflammatory disorder of the gastrointestinal    tract is inflammatory bowel disease.-   (43) The antibody or functional fragment for use according to    item (41) or (42), wherein said inflammatory disorder of the    gastrointestinal tract is Crohn's disease.-   (44) The antibody or functional fragment for use according to item    (43), wherein said Crohn's disease is selected from the group    consisting of ileal, colonic, ileocolonic, and/or isolated upper    Crohn's disease (gastric, duodenal and/or jejunal) and including    non-stricturing/non-penetrating, stricturing, penetrating and    perianal disease behavior, allowing any combination of localization    and disease behavior of any of the above mentioned.-   (45) The antibody or functional fragment for use according to    item (41) or (42), wherein said inflammatory disorder of the    gastrointestinal tract is ulcerative colitis.-   (46) The antibody or functional fragment for use according to item    (45), wherein said ulcerative colitis is selected from the group    consisting of ulcerative proctitis, proctosigmoiditis, left-sided    colitis, pan-ulcerative colitis, and pouchitis.-   (47) The antibody or functional fragment for use according to    item (41) or (42), wherein said inflammatory disorder of the    gastrointestinal tract is microscopic colitis.-   (48) The antibody or functional fragment for use according to any    one of items (39) to (47), wherein said method comprises orally    administering the antibody or functional fragment to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the humanization process.

FIG. 2: SE-HPLC chromatograms of purified humanized scFv preparations ofan scFv. The scFv monomer elutes at retention times between 8.5 and 9.5minutes, while buffer components elute at >10 min. All peaks from thedead volume of the column up to the respective scFv monomer wereintegrated as aggregates/oligomers and used for the calculation of therelative peak area.

FIG. 3: Thermal unfolding curves from DSF measurements of two scFvconstructs. For each construct duplicate measurements are shown. Theresulting Tm values have been determined by fitting the data to aBoltzmann equation to obtain the midpoint of transition.

FIG. 4: Time-course of the monomer content of the two scFv constructsduring storage. The monomer content as determined by SE-HPLC has beenplotted for the storage temperatures 4, −20 and <−65° C. for theduration of 4 weeks.

FIG. 5: Overlay of SE-HPLC chromatograms for two scFv molecules. Foreach scFv the sample (10 mg/ml) at d0 and after storage for 4 weeks at4° C. is shown. In addition, the chromatogram of the sample after 5cycles of freezing and thawing is shown. The inserted panel shows anapprox. 15-fold zoom of the y-axis for each molecule to visualize alsominuscule changes in oligomer content.

FIG. 6: Time-course of the monomer content of the humanized scFvs duringstorage. The monomer content as determined by SE-HPLC has been plottedfor the 10 mg/mL samples at a storage temperature of 37° C. for theduration of 4 weeks.

FIG. 7: Potency to neutralize human TNFα in the L929 assay of two scFvs.Dose-response curves for the scFvs and the reference antibody infliximabare shown for each experiment. The highest scFv and infliximabconcentrations as well as negative controls were set to 100% and 0% ofgrowth.

FIG. 8: Potency of two scFvs to neutralize non-human primate and humanTNFα in the L929 assay. Dose-response curves for neutralization ofhuman, Cynomolgus monkey and Rhesus monkey TNFα are shown. The highestscFv concentration and negative controls were set to 100% and 0% ofgrowth.

FIG. 9: Potency of two scFvs to block the TNFα-TNFRI interaction.Dose-response curves are shown. The highest scFv concentration andnegative controls were set to 0% and 100% of binding of TNFα to TNFRI.

FIG. 10: Potency of two scFvs to block the TNFα-TNFRII interaction.Dose-response curves are shown. The highest scFv concentration andnegative controls were set to 0% and 100% of binding of TNFα to TNFRII.

FIG. 11: Target specificity of an scFv. The potential to inhibit theinteraction of biotinylated TNFα with the scFv by TNFα and TNFβ wasanalyzed by competition ELISA. Dose-dependent effects of TNFα and TNFβare shown.

FIG. 12 depicts the formation of 16-22-H5-scFv:TNFα complexes determinedby SE-HPLC (Example 4).

FIG. 13 depicts the simultaneous binding of two TNFα molecules to16-22-H5-scDb determined by SPR (Example 5).

FIG. 14A depicts the formation of 16-22-H5-IgG:TNFα complexes (Example5).

FIG. 14B depicts the formation of 16-22-H5-scDb:TNFα complexes (Example5).

FIG. 15 depicts the inhibition of cell proliferation in a MLR followinganti-TNFα treatment. *: p<0.05; **: p<0.01 compared to IgG control(Example 6).

FIG. 16 shows the ability of the different antibody formats of 16-22-H5and adalimumab to inhibit the LPS-induced secretion of IL-1β (FIG. 16A)and TNFα (FIG. 16B) in monocytes in a dose-dependent manner (Example 7).

DETAILED DESCRIPTION

The present invention pertains to an antibody or a functional fragmentthereof capable of binding to human TNFα.

In the context of the present application, the term “antibody” is usedas a synonym for “immunoglobulin” (Ig), which is defined as a proteinbelonging to the class IgG, IgM, IgE, IgA, or IgD (or any subclassthereof), and includes all conventionally known antibodies andfunctional fragments thereof. In the context of the present invention, a“functional fragment” of an antibody/immunoglobulin is defined asantigen-binding fragment or other derivative of a parental antibody thatessentially maintains one or more of the properties of such parentalantibody referred to in items (1) to (30) herein above. An“antigen-binding fragment” of an antibody/immunoglobulin is defined asfragment (e.g., a variable region of an IgG) that retains theantigen-binding region. An “antigen-binding region” of an antibodytypically is found in one or more hypervariable region(s) of anantibody, i.e., the CDR-1, -2, and/or -3 regions. “Antigen-bindingfragments” of the invention include the domain of a F(ab′)₂ fragment anda Fab fragment. “Functional fragments” of the invention include, scFv,dsFv, diabodies, triabodies, tetrabodies and Fc fusion proteins. TheF(ab′)₂ or Fab may be engineered to minimize or completely remove theintermolecular disulphide interactions that occur between the CH1 and CLdomains. The antibodies or functional fragments of the present inventionmay be part of bi- or multifunctional constructs.

Preferred functional fragments in the present invention are scFv anddiabodies.

An scFv is a single chain Fv fragment in which the variable light(“V_(L)”) and variable heavy (“V_(H)”) domains are linked by a peptidebridge.

A diabody is a dimer consisting of two fragments, each having variableregions joined together via a linker or the like (hereinafter referredto as diabody-forming fragments), and typically contain two V_(L)s andtwo V_(H)s. Diabody-forming fragments include those consisting of V_(L)and V_(H), V_(L) and V_(L), V_(H) and V_(H), etc., preferably V_(H) andV_(L). In diabody-forming fragments, the linker joining variable regionsis not specifically limited, but preferably enough short to avoidnoncovalent bonds between variable regions in the same fragment.

The length of such a linker can be determined as appropriate by thoseskilled in the art, but typically 2-14 amino acids, preferably 3-9 aminoacids, especially 4-6 amino acids. In this case, the V_(L) and V_(H)encoded on the same fragment are joined via a linker short enough toavoid noncovalent bonds between the V_(L) and V_(H) on the same chainand to avoid the formation of single-chain variable region fragments sothat dimers with another fragment can be formed. The dimers can beformed via either covalent or noncovalent bonds or both betweendiabody-forming fragments.

Moreover, diabody-forming fragments can be joined via a linker or thelike to form single-chain diabodies (sc(Fv)₂). By joiningdiabody-forming fragments using a long linker of about 15-20 aminoacids, noncovalent bonds can be formed between diabody-forming fragmentsexisting on the same chain to form dimers. Based on the same principleas for preparing diabodies, polymerized antibodies such as trimers ortetramers can also be prepared by joining three or more diabody-formingfragments.

Preferably, the antibody or functional fragment of the inventionspecifically binds to TNFα. As used herein, an antibody or functionalfragment thereof “specifically recognizes”, or “specifically binds to”human TNFα, when the antibody or functional fragment is able todiscriminate between human TNFα and one or more reference molecule(s).Preferably, the IC₅₀ value for binding to each of the referencemolecules is at least 1,000 times greater than the IC₅₀ value forbinding to TNFα, particularly as described in Example 2, section 2.1.4.In its most general form (and when no defined reference is mentioned),“specific binding” is referring to the ability of the antibody orfunctional fragment to discriminate between human TNFα and an unrelatedbiomolecule, as determined, for example, in accordance with aspecificity assay methods known in the art. Such methods comprise, butare not limited to, Western blots and ELISA tests. For example, astandard ELISA assay can be carried out. Typically, determination ofbinding specificity is performed by using not a single referencebiomolecule, but a set of about three to five unrelated biomolecules,such as milk powder, BSA, transferrin or the like. In one embodiment,specific binding refers to the ability of the antibody or fragment todiscriminate between human TNFα and human TNFβ.

The antibody of the invention or the functional fragment of theinvention comprises a V_(L) domain and a V_(H) domain. The V_(L) domaincomprises a CDR1 region (CDRL1), a CDR2 region (CDRL2), a CDR3 region(CDRL3) and Framework regions. The V_(H) domain comprises a CDR1 region(CDRH1), a CDR2 region (CDRH2), a CDR3 region (CDRH3) and Frameworkregions.

The term “CDR” refers to one of the six hypervariable regions within thevariable domains of an antibody that mainly contribute to antigenbinding. One of the most commonly used definitions for the six CDRs wasprovided by Kabat E. A. et al., (1991) Sequences of proteins ofimmunological interest. NIH Publication 91-3242). As used herein,Kabat's definition of CDRs only apply for CDR1, CDR2 and CDR3 of thelight chain variable domain (CDR L1, CDR L2, CDR L3, or L1, L2, L3), aswell as for CDR2 and CDR3 of the heavy chain variable domain (CDR H2,CDR H3, or H2, H3). CDR1 of the heavy chain variable domain (CDR H1 orH1), however, as used herein is defined by the following residues (Kabatnumbering): It starts with position 26 and ends prior to position 36.

The CDR1 region of the V_(L) domain consists of an amino acid sequencein accordance with the amino acid sequence as shown in SEQ ID NO:1.Preferably, the CDR1 region of the V_(L) domain consists of an aminoacid sequence selected from the group consisting of SEQ ID NO:7, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21 and SEQ ID NO:22. Most preferably, the CDR1 region of the V_(L)domain consists of the amino acid sequence as shown in SEQ ID NO:7.

The CDR2 region of the V_(L) domain consists of an amino acid sequencein accordance with the amino acid sequence as shown in SEQ ID NO:2.Preferably, the CDR2 region of the V_(L) domain consists of an aminoacid sequence selected from the group consisting of SEQ ID NO:8, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. Most preferably,the CDR2 region of the V_(L) domain consists of the amino acid sequenceas shown in SEQ ID NO:8.

The CDR3 region of the V_(L) domain consists of an amino acid sequencein accordance with the amino acid sequence as shown in SEQ ID NO:3.Preferably, the CDR3 region of the V_(L) domain consists of an aminoacid sequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ IDNO:32. Most preferably, the CDR3 region of the V_(L) domain consists ofthe amino acid sequence as shown in SEQ ID NO:9.

The CDR1 region of the V_(H) domain consists of an amino acid sequencein accordance with the amino acid sequence as shown in SEQ ID NO:4.Preferably, the CDR1 region of the V_(H) domain consists of an aminoacid sequence selected from the group consisting of SEQ ID NO:10, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36. Most preferably, theCDR1 region of the V_(H) domain consists of the amino acid sequence asshown in SEQ ID NO:10.

The CDR2 region of the V_(H) domain consists of an amino acid sequencein accordance with the amino acid sequence as shown in SEQ ID NO:5.Preferably, the CDR2 region of the V_(H) domain consists of an aminoacid sequence selected from the group consisting of SEQ ID NO:11, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41. Mostpreferably, the CDR2 region of the V_(H) domain consists of the aminoacid sequence as shown in SEQ ID NO:11.

The CDR3 region of the V_(H) domain consists of an amino acid sequencein accordance with the amino acid sequence as shown in SEQ ID NO:6.Preferably, the CDR3 region of the V_(H) domain consists of an aminoacid sequence selected from the group consisting of SEQ ID NO:12, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47 and SEQ ID NO:48. Most preferably, the CDR3 region of the V_(H)domain consists of the amino acid sequence as shown in SEQ ID NO:12.

In a particular embodiment, the antibody of the invention or thefunctional fragment of the invention comprises (i) a V_(L) domaincomprising a CDR1 region having an amino acid sequence in accordancewith the amino acid sequence as shown in SEQ ID NO:1, a CDR2 regionhaving an amino acid sequence in accordance with the amino acid sequenceas shown in SEQ ID NO:2, and a CDR3 region having an amino acid sequencein accordance with the amino acid sequence as shown in SEQ ID NO:3, and(ii) a V_(H) domain comprising a CDR1 region having an amino acidsequence in accordance with the amino acid sequence as shown in SEQ IDNO:4, a CDR2 region having an amino acid sequence in accordance with theamino acid sequence as shown in SEQ ID NO:5, and a CDR3 region having anamino acid sequence in accordance with the amino acid sequence as shownin SEQ ID NO:6

In a particular embodiment, the antibody of the invention or thefunctional fragment of the invention comprises (i) a V_(L) domaincomprising a CDR1 region having the amino acid sequence as shown in SEQID NO:7, a CDR2 region having the amino acid sequence as shown in SEQ IDNO:8, and a CDR3 region having the amino acid sequence as shown in SEQID NO:9, and (ii) a V_(H) domain comprising a CDR1 region having theamino acid sequence as shown in SEQ ID NO:10, a CDR2 region having theamino acid sequence as shown in SEQ ID NO:11, and a CDR3 region havingthe amino acid sequence as shown in SEQ ID NO:12.

In a more preferred embodiment, the antibody of the invention or thefunctional fragment of the invention comprises a V_(H) domain having theamino acid sequence as shown in SEQ ID NO:13. In another more preferredembodiment the antibody or functional fragment comprises a V_(L) domainhaving the amino acid sequence as shown in SEQ ID NO:14 or SEQ ID NO:54.Most preferably, the antibody of the invention or the functionalfragment of the invention comprises (i) a V_(H) domain having the aminoacid sequence as shown in SEQ ID NO:13, and (ii) a V_(L) domain havingthe amino acid sequence as shown in SEQ ID NO:14.

In a particularly preferred embodiment, the functional fragment is asingle chain antibody (scFv) comprising a V_(H) domain having the aminoacid sequence as shown in SEQ ID NO:13 and a V_(L) domain having theamino acid sequence as shown in SEQ ID NO:14 or SEQ ID NO:54. The V_(H)domain and the V_(L) domain are preferably linked by a peptide linker.The peptide linker (hereinafter referred to as “linkerA”) typically hasa length of about 10 to about 30 amino acids, more preferably of about15 to about 25 amino acids. The linkerA typically comprises Gly and Serresidues, but other amino acids are also possible. In preferredembodiments the linker comprises multiple repeats of the sequence GGGGS(SEQ ID NO:50), e.g. 2 to 6, or 3 to 5, or 4 consecutive repeats of theamino acid sequence as shown in SEQ ID NO:50. Most preferably, thelinkerA consists of the amino acid sequence as shown in SEQ ID NO:49.The scFv may have the following structure (with the N-terminus beingleft and the C-terminus being right):

V_(L)-LinkerA-V_(H); or

V_(H)-LinkerA-V_(L).

More preferably, the functional fragment is a single chain antibody(scFv) consisting of the amino acid sequence as shown in SEQ ID NO:15 orSEQ ID NO:55. Most preferably, the functional fragment is a single chainantibody (scFv) consisting of the amino acid sequence as shown in SEQ IDNO:15.

In another particularly preferred embodiment, the functional fragment isa diabody comprising a V_(H) domain having the amino acid sequence asshown in SEQ ID NO:13 and a V_(L) domain having the amino acid sequenceas shown in SEQ ID NO:14 or SEQ ID NO:54. The V_(H) domain and the V_(L)domain are linked by a peptide linker. The peptide linker (hereinafterreferred to as “linkerB”) preferably has a length of about 2 to about 10amino acids, more preferably of about 5 amino acids. The linkerBtypically comprises Gly and Ser residues, but other amino acids are alsopossible. Most preferably, the linkerB consists of the amino acidsequence as shown in SEQ ID NO:50.

The diabody preferably is a monospecific diabody, i.e. it is directed toone epitope only. The diabody is preferably a homodimer. The diabody maybe a dimer of two polypeptide chains that are non-covalently bound toeach other. Each monomer may be a polypeptide chain having thestructure:

V_(L)-LinkerB-V_(H); or

V_(H)-LinkerB-V_(L).

Moreover, diabody-forming fragments can be joined via a linkerA or thelike to form single-chain diabodies (sc(Fv)₂). By joiningdiabody-forming fragments using a long linker of about 15-20 aminoacids, noncovalent bonds can be formed between diabody-forming fragmentsexisting on the same chain to form dimers. Examples of the arrangementsof single-chain diabodies include the following.

V_(H)-linkerB-V_(L)-linkerA-V_(H) linkerB—V_(L)

V_(L)-linkerB—V_(H)-linkerA-V_(L)-linkerB—V_(H)

Preferably the diabody of the invention has the following structure:

V_(L)-linkerB—V_(H)-linkerA-V_(L)-linkerB—V_(H)

Most preferably the diabody consists of the amino acid sequence as shownin SEQ ID NO:51.

Based on the same principle as for preparing diabodies, polymerizedantibodies such as trimers or tetramers can also be prepared by joiningthree or more diabody-forming fragments.

In another particular embodiment the antibody of the invention is animmunoglobulin, preferably an immunoglobulin G (IgG). The subclass ofthe IgG of the invention is not limited and includes IgG₁, IgG₂, IgG₃,and IgG₄. Preferably, the IgG of the invention is of subclass 1, i.e. itis an IgG, molecule. In one embodiment, each light chain of the IgGmolecule of the invention has the amino acid sequence as shown in SEQ IDNO:52, and/or each heavy chain of the IgG molecule of the invention hasthe amino acid sequence as shown in SEQ ID NO:53. A specific IgG of theinvention consists of two light chains and two heavy chains, whereineach of the two light chains has the amino acid sequence as shown in SEQID NO:52, and each of the two heavy chains has the amino acid sequenceas shown in SEQ ID NO:53.

Affinity

The antibody or functional fragment of the invention has a high affinityto human TNFα. The term “K_(D),” refers to the dissociation equilibriumconstant of a particular antibody-antigen interaction. Typically, theantibody or functional fragment of the invention binds to human TNFαwith a dissociation equilibrium constant (K_(D)) of less thanapproximately 2×10⁻¹° M, preferably less than 1.5×10⁻¹° M, preferablyless than 1.25×10⁻¹° M, more preferably less than 1×10⁻¹⁰ M, mostpreferably less than 7.5×10⁻¹¹ M or even less than 5×10⁻¹¹ M, asdetermined using surface plasmon resonance (SPR) technology in a BIACOREinstrument. In particular, the determination of the K_(D) is carried outas described in Example 2, section 2.1.1.

Cross-Reactivity to TNFα from Cynomolgus Monkeys or from Rhesus Macaques

In particular embodiments, the antibody or functional fragment of theinvention has substantial affinity to TNFα from animals such asCynomolgus monkeys (Macaca fascicularis) and/or Rhesus macaques (Macacamulatta). This is advantageous, as preclinical tests of anti-human TNFαantibodies such as toxicity studies are preferably performed with suchanimals. Accordingly, the antibody or functional fragment of theinvention is preferably cross-reactive with TNFα from animals such asCynomolgus monkeys and/or Rhesus macaques. Affinity measurements arecarried out as described in Example 2, section 2.1.1.

In one embodiment, the antibody or functional fragment of the inventionis cross-reactive with TNFα from Macaca fascicularis. The antibody orfunctional fragment of the invention preferably has an affinity toMacaca fascicularis TNFα that is less than 20-fold, particularly lessthan 10-fold, even more particularly less than 5-fold different to thatof human TNFα. Typically, the antibody or functional fragment of theinvention binds to TNFα from Macaca fascicularis with a dissociationequilibrium constant (K_(D)), wherein the ratio R_(M. fascicularis) of(i) the K_(D) for binding to TNFα from Macaca fascicularis to (ii) theK_(D) for binding to human TNFα is less than 20.

$R_{M.{fascicularis}.} = \frac{K_{D}\left( {{M.{fasc}}{icularis}} \right)}{K_{D}({human})}$

R_(M. fascicularis) is preferably less than 20, particularly less than10, even more particularly less than 5.

In another embodiment, the antibody or functional fragment of theinvention is cross-reactive with TNFα from Macaca mulatta. The antibodyor functional fragment of the invention preferably has an affinity toMacaca mulatta TNFα that is less than 20-fold, more particularly lessthan 10-fold different to that of human TNFα. Typically, the antibody orfunctional fragment of the invention binds to TNFα from Macaca mulattawith a dissociation equilibrium constant (K_(D)), wherein the ratioR_(m) mulatta of (i) the K_(D) for binding to TNFα from Macaca mulattato (ii) the K_(D) for binding to human TNFα is less than 20.

$R_{{M.{mul}}atta} = \frac{K_{D}\left( {{M.m}ulatta} \right)}{K_{D}({human})}$

R_(M.) mulatta is preferably less than 20, particularly less than 10.

In yet another embodiment, the antibody or functional fragment of theinvention is cross-reactive with TNFα from Macaca fascicularis and withTNFα from Macaca mulatta. The antibody or functional fragment of theinvention preferably has an affinity to Macaca fascicularis TNFα that isless than 20-fold, particularly less than 10-fold, even moreparticularly less than 5-fold different to that of human TNFα, and itpreferably has an affinity to Macaca mulatta TNFα that is less than20-fold, more particularly less than 10-fold different to that of humanTNFα. The ratio R_(M. fasciculans) of the antibody or functionalfragment is preferably less than 20, particularly less than 10, evenmore particularly less than 5, and the ratio R_(M. mulatta) of theantibody or functional fragment is preferably less than 20, particularlyless than 10.

Potency to Inhibit TNFα-Induced Apoptosis of L929 Cells

The antibody or functional fragment of the invention has a high potencyto inhibit TNFα-induced apoptosis of L929 cells. In a particularembodiment, the antibody or functional fragment of the invention has apotency to inhibit TNFα-induced apoptosis of L929 cells greater thanthat of the known antibody infliximab.

Potency relative to infliximab can be determined in an L929 assay asdescribed in Example 2, section 2.1.2 of this application. The relativepotency of the antibody or functional fragment of the invention isgreater than 1.5, preferably greater than 2, more preferably greaterthan 3, more preferably greater than 5, more preferably greater than7.5, or even greater than 10, wherein the relative potency is the ratioof (i) the IC₅₀ value of infliximab in an L929 assay over (ii) the IC₅₀value of the antibody or functional fragment of the invention in theL929 assay, and wherein the IC₅₀ indicates the concentration in ng/mL ofthe respective molecule necessary to achieve 50% of maximal inhibitionof TNFα-induced apoptosis of L929 cells.

In another embodiment, the relative potency of the antibody orfunctional fragment of the invention is greater than 1.5, preferablygreater than 2, more preferably greater than 3, more preferably greaterthan 5, more preferably greater than 7.5, or even greater than 10,wherein the relative potency is the ratio of (i) the IC₉₀ value ofinfliximab in an L929 assay over (ii) the IC₉₀ value of the antibody orfunctional fragment of the invention in the L929 assay, and wherein theIC₉₀ value indicates the concentration in ng/mL of the respectivemolecule necessary to achieve 90% of maximal inhibition of TNFα-inducedapoptosis of L929 cells.

Inhibition of LPS-Induced Cytokine Secretion

Typically, the antibody or functional fragment of the invention iscapable of inhibiting LPS-induced cytokine secretion from monocytes.LPS-induced cytokine secretion from monocytes can be determined asdescribed in Example 7.

In one embodiment, the antibody or functional fragment of the inventionis capable of inhibiting LPS-induced secretion of interleukin-1β fromCD14⁺ monocytes. The IC₅₀ value for inhibiting LPS-induced secretion ofinterleukin-1β is preferably less than 1 nM and/or less than 100 pg/mL.The IC₅₀ value for inhibiting LPS-induced secretion of interleukin-1β,on a molar basis and/or on a weight-per-volume basis, is preferablylower than that of adalimumab.

In another embodiment, the antibody or functional fragment of theinvention is capable of inhibiting LPS-induced secretion of TNFα fromCD14⁺ monocytes. The IC₅₀ value for inhibiting LPS-induced secretion ofTNFα is preferably less than 1 nM and/or less than 150 pg/mL. The IC₅₀value for inhibiting LPS-induced secretion of TNFα, on a molar basisand/or on a weight-per-volume basis, is preferably lower than that ofadalimumab.

Inhibition of Cell Proliferation

The antibody or functional fragment of the invention is typicallycapable of inhibiting cell proliferation of peripheral blood mononuclearcells in a mixed lymphocyte reaction. The inhibition of cellproliferation can be determined as described in Example 6. Thestimulation index of the antibody or functional fragment, e.g. of thescFv or diabody of the invention, determined according to Example 6, ispreferably less than 5, more preferably less than 4.5. In particularembodiments, the stimulation index of the antibody, e.g. of the IgG ofthe invention, is less than 4 or even less than 3.

Inhibition of Interaction Between TNFα and TNF Receptor

Typically, the antibody or functional fragment of the invention iscapable of inhibiting the interaction between human TNFα and TNFreceptor I (TNFRI). The inhibition of the interaction between human TNFαand TNFRI can be determined in an inhibition ELISA as described below inExample 2, section 2.1.3.

The potency of the antibody or functional fragment of the invention toinhibit the interaction between human TNFα and TNFRI, relative to thatof infliximab (relative potency), as determined in an inhibition ELISA,is preferably at least 2, wherein said relative potency is the ratio ofthe 10₅₀ value in ng/mL of infliximab to the 10₅₀ value in ng/mL of theantibody or functional fragment thereof.

Typically, the antibody or functional fragment of the invention iscapable of inhibiting the interaction between human TNFα and TNFreceptor II (TNFRII). The inhibition of the interaction between humanTNFα and TNFRII can be determined in an inhibition ELISA as describedbelow in Example 2, section 2.1.3.

The potency of the antibody or functional fragment of the invention toinhibit the interaction between human TNFα and TNFRII, relative to thatof infliximab (relative potency), as determined in an inhibition ELISA,is preferably at least 2, more preferably at least 3, wherein saidrelative potency is the ratio of the IC₅₀ value in ng/mL of infliximabto the IC₅₀ value in ng/mL of the antibody or functional fragmentthereof.

Stoichiometry and Crosslinking

The antibody or functional fragment of the invention is typicallycapable of binding to human TNFα_(Trimer) in a stoichiometry (antibody:TNFα_(Trimer)) of at least 2. The stoichiometry (antibody:TNFα_(Trimer)) is preferably greater than 2, or at least 2.5, or atleast 3. In one embodiment, the stoichiometry (antibody: TNFα_(Trimer))is about 3. The stoichiometry (antibody: TNFα_(Trimer)) can bedetermined as described in Example 4 below.

In another embodiment, the antibody or functional fragment of theinvention is capable of forming a complex with human TNFα, wherein saidcomplex comprises at least two molecules of TNFα and at least threemolecules of antibody or functional fragment. The functional fragment inaccordance with this embodiment comprises at least two separate bindingsites for TNFα such as, e.g. diabodies. Complex formation can bedetermined as described in Example 5 below.

In one embodiment, the antibody is an IgG, and is capable of forming acomplex of at least 600 kDa with TNFα. In another embodiment, thefunctional fragment is a diabody, and is capable of forming a complex ofat least 300 kDa with TNFα.

Target Selectivity

In certain embodiments, the antibody or the functional fragment of theinvention has a high target selectivity, i.e. it can discriminatebetween TNFα and TNFβ. Preferably, the IC₅₀ value of TNFβ is at least1,000 times greater than the IC₅₀ value of TNFα, as determined in acompetition ELISA as described in Example 2, section 2.1.4. Morepreferably, the IC₅₀ value of TNFβ is at least 5,000 times, mostpreferably at least 10,000 greater than the IC₅₀ value of TNFα, asdetermined in a competition ELISA as described in Example 2, section2.1.4.

Expression Yield and Refolding Yield

In other embodiments, the antibody or functional fragment of theinvention, preferably the scFv or diabody, can be recombinantlyexpressed in high yield in microorganisms such as bacteria or in othercells. Preferably, the expression yield in E. coli, determined asdescribed in Example 2, is at least 0.25 g/L. This particularly appliesto functional fragments such as scFvs.

The refolding yield, determined as described in Example 2, is at least 5mg/L, more preferably at least 10 mg/L, more preferably at least 15mg/L, and most preferably at least 20 mg/L. This particularly applies tofunctional fragments such as scFvs.

Stability

Typically, the antibody or functional fragment of the invention,preferably the scFv or diabody, has a high stability. Stability can beassessed by different methodologies. The “melting temperature” T_(m) ofthe variable domain of the antibody or functional fragment of theinvention, determined by differential scanning fluorimetry (DSF) asdescribed in Example 2, section 2.2.4, is preferably at least 65° C.,more preferably at least 68° C., most preferably at least 70° C. The“melting temperature of the variable domain”, as used herein, refers tothe melting temperature of an scFv consisting of the sequenceV_(L)-LinkerA-V_(H), wherein the amino acid sequence of LinkerA consistsof the amino acid sequence as shown in SEQ ID NO:49. For example, themelting temperature of the variable domain of an IgG is defined as themelting temperature of its corresponding scFv as defined above.

The loss in monomer content (at a concentration of 10 g/L; initialmonomer content >95%) after storage for four weeks at 4° C., determinedby analytical size-exclusion chromatography as described in Example 2,section 2.2.5, is preferably less than 5%, more preferably less than 3%,more preferably less than 1%, most preferably less than 0.5%. The lossin monomer content (at a concentration of 10 g/L; initial monomercontent >95%) after storage for four weeks at −20° C., determined byanalytical size-exclusion chromatography as described in Example 2,section 2.2.5, is preferably less than 5%, more preferably less than 3%,more preferably less than 1%, most preferably less than 0.5%. The lossin monomer content (at a concentration of 10 g/L; initial monomercontent >95%) after storage for four weeks at −65° C., determined byanalytical size-exclusion chromatography as described in Example 2,section 2.2.5, is preferably less than 5%, more preferably less than 3%,more preferably less than 1%, most preferably less than 0.5%.

The monomer loss after five consecutive freeze-thaw cycles, determinedas described in Example 2, is less than 5%, more preferably less than1%, more preferably less than 0.5%, most preferably less than 0.2%, e.g.0.1% or 0.0%.

Antibodies and Functional Fragments

Particular embodiments of the invention relate to functional fragmentsof the antibodies described herein. Functional fragments include, butare not limited to, F(ab′)₂ fragment, a Fab fragment, scFv, diabodies,triabodies and tetrabodies. Preferably, the functional fragment is asingle chain antibody (scFv) or a diabody. More preferably, the non-CDRsequences of the scFv or of the diabody are human sequences.

Preferably in order to minimize potential for immunogenicity in humansthe chosen acceptor scaffold is composed of framework regions derivedfrom human consensus sequences or human germline sequences. Inparticular framework regions I to III of the variable light domainconsist of human VK1 consensus sequences according to SEQ ID NOs: 56 to58 and a framework region IV of a A germline-based sequence selectedfrom SEQ ID NOs:59 to 62. As residues that are not human consensus orhuman germline residues may cause immune reactions the number of suchresidues in each variable domain (VH or VL) should be as low aspossible, preferably lower than 7, more preferably lower than 4, mostpreferably 0.

Preferably the antibody is a monoclonal antibody. The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. (Harlow and Lane,“Antibodies, A Laboratory Manual” CSH Press 1988, Cold Spring HarborN.Y.).

In other embodiments, including embodiments relating to the in vivo useof the anti-TNFα antibodies in humans, chimeric, primatized, humanized,or human antibodies can be used. In a preferred embodiment, the antibodyis a human antibody or a humanized antibody, more preferably amonoclonal human antibody or a monoclonal humanized antibody.

The term “chimeric” antibody as used herein refers to an antibody havingvariable sequences derived from a non-human immunoglobulin, such as arat or mouse antibody, and human immunoglobulins constant regions,typically chosen from a human immunoglobulin template. Methods forproducing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science 229(4719): 1202-7; Oi et al, 1986, BioTechniques4:214-221; Gillies et al., 1985, J. Immunol. Methods 125: 191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entireties.

Different recombinant methodologies are available to one of ordinaryskill in the art to render a non-human (e.g., murine) antibody morehuman-like by generating immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or othertarget-binding subsequences of antibodies), which contain minimalsequences derived from such non-human immunoglobulin. In general, theresulting recombinant antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence, particularly a human immunoglobulinconsensus sequence. CDR-grafted antibodies are antibody molecules havingone or more complementarity determining regions (CDRs) from an antibodyoriginally generated in a non-human species that bind the desiredantigen and framework (FR) regions from a human immunoglobulin molecule(EP239400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539;5,530,101 and 5,585,089). Often, in a process called “humanization”,framework residues in the human framework regions will additionally besubstituted with the corresponding residue from the CDR donor antibodyto alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. See, e.g., Riechmann et al., 1988, Nature 332:323-7 and Queenet al, U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and6,180,370 (each of which is incorporated by reference in its entirety).Antibodies can be rendered more human using a variety of additionaltechniques known in the art including, for example, veneering orresurfacing (EP592106; EP519596; Padlan, 1991, Mol. Immunol, 28:489-498;Studnicka et al, 1994, Prot. Eng. 7:805-814; Roguska et al, 1994, Proc.Natl. Acad. Sci. 91:969-973, and chain shuffling (U.S. Pat. No.5,565,332), all of which are hereby incorporated by reference in theirentireties. A CDR-grafted or humanized antibody can also comprise atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin template chosen.

In some embodiments, humanized antibodies are prepared as described inQueen et al, U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762;and 6,180,370 (each of which is incorporated by reference in itsentirety).

In some embodiments, the anti-TNFα antibodies are human antibodies.Completely “human” anti-TNFα antibodies can be desirable for therapeutictreatment of human patients. As used herein, “human antibodies” includeantibodies having the amino acid sequence of a human immunoglobulin andinclude antibodies isolated from human immunoglobulin libraries or fromanimals transgenic for one or more human immunoglobulin and that do notexpress endogenous immunoglobulins. Human antibodies can be made by avariety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111;and PCT publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654;WO 96/34096; WO 96/33735; and WO 91/10741, each of which is incorporatedherein by reference in its entirety. Human antibodies can also beproduced using transgenic mice which are incapable of expressingfunctional endogenous immunoglobulins, but which can express humanimmunoglobulin genes. See, e.g., PCT publications WO 98/24893; WO92/01047; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. Completely human antibodies that recognize a selectedepitope can be generated using a technique referred to as “guidedselection.” In this approach a selected non-human monoclonal antibody,e.g., a mouse antibody, is used to guide the selection of a completelyhuman antibody recognizing the same epitope (Jespers et al, 1988,Biotechnology 12:899-903).

In some embodiments, the anti-TNFα antibodies are primatized antibodies.The term “primatized antibody” refers to an antibody comprising monkeyvariable regions and human constant regions. Methods for producingprimatized antibodies are known in the art. See e.g., U.S. Pat. Nos.5,658,570; 5,681,722; and 5,693,780, which are incorporated herein byreference in their entireties.

In some embodiments, the anti-TNFα antibodies are derivatizedantibodies. For example, but not by way of limitation, the derivatizedantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein (see below for a discussion of antibodyconjugates), etc. Any of numerous chemical modifications may be carriedout by known techniques, including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc. Additionally, the derivative may contain one or morenon-classical amino acids.

In yet other aspects, an anti-TNFα antibody has one or more amino acidsinserted into one or more of its hypervariable region, for example asdescribed in US 2007/0280931.

Antibody Conjugates

In some embodiments, the anti-TNFα antibodies are antibody conjugatesthat are modified, e.g., by the covalent attachment of any type ofmolecule to the antibody, such that covalent attachment does notinterfere with binding to TNFα. Techniques for conjugating effectormoieties to antibodies are well known in the art (See, e.g., Hellstromet al., Controlled Drag Delivery, 2nd Ed., at pp. 623-53 (Robinson etal., eds., 1987)); Thorpe et al., 1982, Immunol. Rev. 62: 119-58 andDubowchik et al., 1999, Pharmacology and Therapeutics 83:67-123).

In one example, the antibody or fragment thereof is fused via a covalentbond (e.g., a peptide bond), at optionally the N-terminus or theC-terminus, to an amino acid sequence of another protein (or portionthereof; preferably at least a 10, 20 or 50 amino acid portion of theprotein). Preferably the antibody, or fragment thereof, is linked to theother protein at the N-terminus of the constant domain of the antibody.Recombinant DNA procedures can be used to create such fusions, forexample as described in WO 86/01533 and EP0392745. In another examplethe effector molecule can increase half-life in vivo. Examples ofsuitable effector molecules of this type include polymers, albumin,albumin binding proteins or albumin binding compounds such as thosedescribed in WO 2005/117984.

In some embodiments, anti-TNFα antibodies can be attached topoly(ethyleneglycol) (PEG) moieties. For example, if the antibody is anantibody fragment, the PEG moieties can be attached through anyavailable amino acid side-chain or terminal amino acid functional grouplocated in the antibody fragment, for example any free amino, imino,thiol, hydroxyl or carboxyl group. Such amino acids can occur naturallyin the antibody fragment or can be engineered into the fragment usingrecombinant DNA methods. See for example U.S. Pat. No. 5,219,996.Multiple sites can be used to attach two or more PEG molecules.Preferably PEG moieties are covalently linked through a thiol group ofat least one cysteine residue located in the antibody fragment. Where athiol group is used as the point of attachment, appropriately activatedeffector moieties, for example thiol selective derivatives such asmaleimides and cysteine derivatives, can be used.

In another example, an anti-TNFα antibody conjugate is a modified Fab′fragment which is PEGylated, i.e., has PEG (poly(ethyleneglycol))covalently attached thereto, e.g., according to the method disclosed inEP0948544. See also Poly(ethyleneglycol) Chemistry, Biotechnical andBiomedical Applications, (J. Milton Harris (ed.), Plenum Press, NewYork, 1992); Poly(ethyleneglycol) Chemistry and Biological Applications,(J. Milton Harris and S. Zalipsky, eds., American Chemical Society,Washington D. C, 1997); and Bioconjugation Protein Coupling Techniquesfor the Biomedical Sciences, (M. Aslam and A. Dent, eds., GrovePublishers, New York, 1998); and Chapman, 2002, Advanced Drug DeliveryReviews 54:531-545.

Pharmaceutical Compositions and Treatment

Treatment of a disease encompasses the treatment of patients alreadydiagnosed as having any form of the disease at any clinical stage ormanifestation; the delay of the onset or evolution or aggravation ordeterioration of the symptoms or signs of the disease; and/or preventingand/or reducing the severity of the disease.

A “subject” or “patient” to whom an anti-TNFα antibody or functionalfragment thereof is administered can be a mammal, such as a non-primate(e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkeyor human). In certain aspects, the human is a pediatric patient. Inother aspects, the human is an adult patient.

Compositions comprising an anti-TNFα antibody and, optionally one ormore additional therapeutic agents, such as the second therapeuticagents described below, are described herein. The compositions typicallyare supplied as part of a sterile, pharmaceutical composition thatincludes a pharmaceutically acceptable carrier. This composition can bein any suitable form (depending upon the desired method of administeringit to a patient).

The anti-TNFα antibodies and functional fragments can be administered toa patient by a variety of routes such as orally, transdermally,subcutaneously, intranasally, intravenously, intramuscularly,intrathecally, topically or locally. The most suitable route foradministration in any given case will depend on the particular antibody,the subject, and the nature and severity of the disease and the physicalcondition of the subject. Typically, an anti-TNFα antibody or functionalfragment thereof will be administered intravenously.

In a particularly preferred embodiment, the antibody or functionalfragment of the invention is administered orally. If the administrationis via the oral route the functional fragment is preferably a singlechain antibody (scFv), diabody or IgG.

In typical embodiments, an anti-TNFα antibody or functional fragment ispresent in a pharmaceutical composition at a concentration sufficient topermit intravenous administration at 0.5 mg/kg body weight to 20 mg/kgbody weight. In some embodiments, the concentration of antibody orfragment suitable for use in the compositions and methods describedherein includes, but is not limited to, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg,2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, or aconcentration ranging between any of the foregoing values, e.g., 1 mg/kgto 10 mg/kg, 5 mg/kg to 15 mg/kg, or 10 mg/kg to 18 mg/kg.

The effective dose of an anti-TNFα antibody or functional fragment canrange from about 0.001 to about 750 mg/kg per single (e.g., bolus)administration, multiple administrations or continuous administration,or to achieve a serum concentration of 0.01-5000 μg/ml serumconcentration per single (e.g., bolus) administration, multipleadministrations or continuous administration, or any effective range orvalue therein depending on the condition being treated, the route ofadministration and the age, weight and condition of the subject. Incertain embodiments, each dose can range from about 0.5 mg to about 50mg per kilogram of body weight or from about 3 mg to about 30 mg perkilogram body weight. The antibody can be formulated as an aqueoussolution.

Pharmaceutical compositions can be conveniently presented in unit doseforms containing a predetermined amount of an anti-TNFα antibody orfunctional fragment per dose. Such a unit can contain 0.5 mg to 5 g, forexample, but without limitation, 1 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 750 mg, 1000 mg, or anyrange between any two of the foregoing values, for example 10 mg to 1000mg, 20 mg to 50 mg, or 30 mg to 300 mg. Pharmaceutically acceptablecarriers can take a wide variety of forms depending, e.g., on thecondition to be treated or route of administration.

Determination of the effective dosage, total number of doses, and lengthof treatment an anti-TNFα antibody or functional fragment thereof iswell within the capabilities of those skilled in the art, and can bedetermined using a standard dose escalation study.

Therapeutic formulations of the anti-TNFα antibodies and functionalfragments suitable in the methods described herein can be prepared forstorage as lyophilized formulations or aqueous solutions by mixing theantibody having the desired degree of purity with optionalpharmaceutically-acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are referred to herein as“carriers”), i.e., buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants, and othermiscellaneous additives. See, Remington's Pharmaceutical Sciences, 16thedition (Osol, ed. 1980). Such additives must be nontoxic to therecipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They can present at concentration ranging fromabout 2 mM to about 50 mM. Suitable buffering agents include bothorganic and inorganic acids and salts thereof such as citrate buffers(e.g., monosodium citrate-disodium citrate mixture, citricacid-trisodium citrate mixture, citric acid-monosodium citrate mixture,etc.), succinate buffers (e.g., succinic acid-monosodium succinatemixture, succinic acid-sodium hydroxide mixture, succinic acid-disodiumsuccinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodiumtartrate mixture, tartaric acid-potassium tartrate mixture, tartaricacid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaricacid-monosodium fumarate mixture, fumaric acid-disodium fumaratemixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconatebuffers (e.g., gluconic acid-sodium glyconate mixture, gluconicacid-sodium hydroxide mixture, gluconic acid-potassium glyuconatemixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalatemixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassiumoxalate mixture, etc), lactate buffers (e.g., lactic acid-sodium lactatemixture, lactic acid-sodium hydroxide mixture, lactic acid-potassiumlactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodiumacetate mixture, acetic acid-sodium hydroxide mixture, etc.).Additionally, phosphate buffers, histidine buffers and trimethylaminesalts such as Tris can be used.

Preservatives can be added to retard microbial growth, and can be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives includephenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben,octadecyldimethylbenzyl ammonium chloride, benzalconium halides (e.g.,chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens such as methyl or propyl paraben, catechol, resorcinol,cyclohexanol, and 3-pentanol. Isotonicifiers sometimes known as“stabilizers” can be added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Stabilizers refer to a broad category ofexcipients which can range in function from a bulking agent to anadditive which solubilizes the therapeutic agent or helps to preventdenaturation or adherence to the container wall. Typical stabilizers canbe polyhydric sugar alcohols (enumerated above); amino acids such asarginine, lysine, glycine, glutamine, asparagine, histidine, alanine,ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.,organic sugars or sugar alcohols, such as lactose, trehalose, stachyose,mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glyceroland the like, including cyclitols such as inositol; polyethylene glycol;amino acid polymers; sulfur containing reducing agents, such as urea,glutathione, thioctic acid, sodium thioglycolate, thioglycerol,α-monothioglycerol and sodium thio sulfate; low molecular weightpolypeptides (e.g., peptides of 10 residues or fewer); proteins such ashuman serum albumin, bovine serum albumin, gelatin or immunoglobulins;hydrophylic polymers, such as polyvinylpyrrolidone monosaccharides, suchas xylose, mannose, fructose, glucose; disaccharides such as lactose,maltose, sucrose and trisaccacharides such as raffinose; andpolysaccharides such as dextran. Stabilizers can be present in the rangefrom 0.1 to 10,000 weights per part of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) canbe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20,TWEEN®-80, etc.). Non-ionic surfactants can be present in a range ofabout 0.05 mg/ml to about 1.0 mg/ml, or in a range of about 0.07 mg/mlto about 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), protease inhibitors and co-solvents.

The formulation herein can also contain a second therapeutic agent inaddition to an anti-TNFα antibody or functional fragment thereof.Examples of suitable second therapeutic agents are provided below.

The dosing schedule can vary from once a month to daily depending on anumber of clinical factors, including the type of disease, severity ofdisease, and the patient's sensitivity to the anti-TNFα antibody orfunctional fragment. In specific embodiments, an anti-TNFα antibody orfunctional fragment thereof is administered daily, twice weekly, threetimes a week, every other day, every 5 days, every 10 days, every twoweeks, every three weeks, every four weeks or once a month, or in anyrange between any two of the foregoing values, for example from everyfour days to every month, from every 10 days to every two weeks, or fromtwo to three times a week, etc.

The dosage of an anti-TNFα antibody or functional fragment to beadministered will vary according to the particular antibody, thesubject, and the nature and severity of the disease, the physicalcondition of the subject, the therapeutic regimen (e.g., whether asecond therapeutic agent is used), and the selected route ofadministration; the appropriate dosage can be readily determined by aperson skilled in the art.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of an anti-TNFα antibody orfunctional fragment thereof will be determined by the nature and extentof the condition being treated, the form, route and site ofadministration, and the age and condition of the particular subjectbeing treated, and that a physician will ultimately determineappropriate dosages to be used. This dosage can be repeated as often asappropriate. If side effects develop the amount and/or frequency of thedosage can be altered or reduced, in accordance with normal clinicalpractice.

Disorders to be Treated

The invention relates to a method of treating or preventing a humanTNFα-related disease in a subject, comprising administering to thesubject the antibody or functional fragment as defined herein. The term“TNFα-related disorder” or “TNFα-related disease” refers to anydisorder, the onset, progression or the persistence of the symptoms ordisease states of which requires the participation of TNFα. ExemplaryTNFα-related disorders include, but are not limited to, chronic and/orautoimmune states of inflammation in general, immune mediatedinflammatory disorders in general, inflammatory CNS disease,inflammatory diseases affecting the eye, joint, skin, mucous membranes,central nervous system, gastrointestinal tract, urinary tract or lung,states of uveitis in general, retinitis, HLA-B27+uveitis, Behçet'sdisease, dry eye syndrome, glaucoma, Sjögren syndrome, diabetes mellitus(incl. diabetic neuropathy), insulin resistance, states of arthritis ingeneral, rheumatoid arthritis, osteoarthritis, reactive arthritis andReiter's syndrome, juvenile arthritis, ankylosing spondylitis, multiplesclerosis, Guillain-Barré syndrome, myasthenia gravis, amyotrophiclateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidneydisease, cystitis, psoriasis (incl. psoriatic arthritis), hidradenitissuppurativa, panniculitis, pyoderma gangrenosum, SAPHO syndrome(synovitis, acne, pustulosis, hyperostosis and osteitis), acne, Sweet'ssydrome, pemphigus, Crohn's disease (incl. extraintestinalmanifestastations), ulcerative colitis, asthma bronchiale,hypersensitivity pneumonitis, general allergies, allergic rhinitis,allergic sinusitis, chronic obstructive pulmonary disease (COPD), lungfibrosis, Wegener's granulomatosis, Kawasaki syndrome, Giant cellarteritis, Churg-Strauss vasculitis, polyarteritis nodosa, burns, graftversus host disease, host versus graft reactions, rejection episodesfollowing organ or bone marrow transplantation, systemic and localstates of vasculitis in general, systemic and cutaneous lupuserythematodes, polymyositis and dermatomyositis, sclerodermia,pre-eclampsia, acute and chronic pancreatitis, viral hepatitis,alcoholic hepatitis, postsurgical inflammation such as after eye surgery(e.g. cataract (eye lens replacement) or glaucoma surgery), jointsurgery (incl. arthroscopic surgery), surgery at joint-relatedstructures (e.g. ligaments), oral and/or dental surgery, minimallyinvasive cardiovascular procedures (e.g. PTCA, atherectomy, stentplacement), laparoscopic and/or endoscopic intra-abdominal andgynecological procedures, endoscopic urological procedures (e.g.prostate surgery, ureteroscopy, cystoscopy, interstitial cystitis), orperioperative inflammation (prevention) in general, bullous dermatitis,neutrophilic dermatitis, toxic epidermal necrolysis, pustulardermatitis, cerebral malaria, hemolytic uremic syndrome, allograftrejection, otitis media, snakebite, erythema nodosum, myelodysplasticsyndromes, primary sclerosing cholangitis, seronegativespondylartheropathy, autoimmune hematolytic anemia, orofacialgranulamatosis, pyostomatitis vegetans, aphthous stomatitis, geographictongue, migratory stoimatitis, Alzheimer disease, Parkinson's disease,Huntington's disease, Bell's palsy, Creutzfeld-Jakob disease andneuro-degenerative conditions in general.

Cancer-related osteolysis, cancer-related inflammation, cancer-relatedpain, cancer-related cachexia, bone metastases, acute and chronic formsof pain, irrespective whether these are caused by central or peripheraleffects of TNFα and whether they are classified as inflammatory,nociceptive or neuropathic forms of pain, sciatica, low back pain,carpal tunnel syndrome, complex regional pain syndrome (CRPS), gout,postherpetic neuralgia, fibromyalgia, local pain states, chronic painsyndroms due to metastatic tumor, dismenorrhea.

Particular disorders to be treated include states of arthritis ingeneral, rheumatoid arthritis, osteoarthritis, reactive arthritis,juvenile arthritis; psoriasis incl. psoriatic arthritis; inflammatorybowel disease, including Crohn's disease, ulcerative colitis incl.proctitis, sigmoiditis, left-sided colitis, extensive colitis andpancolitis, undetermined colitis, microscopic colitis incl. collagenousand lymphocytic colitis, colitis in connective tissue disease, diversioncolitis, colitis in diverticular disease, eosinophilic colitis andpouchitis.

Most preferably, the antibody or functional fragment of the invention isused to treat an inflammatory bowel disease, in particular Crohn'sdisease, ulcerative colitis or microscopic colitis. The Crohn's diseasemay be ileal, colonic, ileocolonic or isolated upper Crohn's disease(gastric, duodenal and/or jejunal) includingnon-stricturing/non-penetrating, stricturing, penetrating and perianaldisease behavior, allowing any combination of localization and diseasebehavior of any of the above mentioned. The ulcerative colitis may beulcerative proctitis, proctosigmoiditis, left-sided colitis,pan-ulcerative colitis and pouchitis.

Combination Therapy and Other Aspects

Preferably, the patient being treated with an anti-TNFα antibody orfunctional fragment thereof is also treated with another conventionalmedicament. For example, a patient suffering from inflammatory boweldisease, especially if having moderate to severe disease is typicallyalso being treated with mesalazine or derivatives or prodrugs thereof,corticosteroids, e.g. budesonide or prednisolone (oral or i.v.),immunosuppressants, e.g. azathioprine/6-mercaptopurine (6-MP) ormethotrexate, cyclosporine or tacrolimus. Other medicaments which can beco-administered to the patient include biologics such as infliximab,adalimumab, etanercept, certolizumab pegol or others. Furthermedicaments which can be co-administered to the patient includeimmunosupressants (e.g. azathioprine/6-MP or methotrexate or oralcyclosporine) in order to maintain stable and longer remission. Yetanother aspect of the invention is the use of an anti-TNFα antibody orfunctional fragment as defined hereinabove for reducing inflammation.

Yet another aspect of the invention is an anti-TNFα antibody orfunctional fragment as defined hereinabove for use in reducinginflammation in a patient suffering from an inflammatory condition.

A further aspect of this invention is a method of treating aninflammatory condition, comprising administering to a patient in needthereof an effective amount of an anti-TNFα antibody or functionalfragment as defined hereinabove. The inflammatory condition ispreferably one of the conditions described above.

A further aspect of this invention is a method of preventing aninflammatory condition, comprising administering to a patient in needthereof an effective amount of an anti-TNFα antibody or functionalfragment as defined hereinabove. The inflammatory condition ispreferably one of the conditions described above.

TABLE 1 Summary of the amino acid sequences SEQ ID NO: Description 1Generic CDR L1 2 Generic CDR L2 3 Generic CDR L3 4 Generic CDR H1 5Generic CDR H2 6 Generic CDR H3 7 CDR L1 of clone 16-22-H05 8 CDR L2 ofclone 16-22-H05 and clone 16-16-H10 9 CDR L3 of clone 16-22-H05 andclone 16-16-H10 10 CDR H1 of clone 16-22-H05, clone 16-16-B08 and clone16-16-H10 11 CDR H2 of clone 16-22-H05 12 CDR H3 of clone 16-22-H05 13V_(H) of humanized scFv of clone 16-22-H05-sc02 and clone 16-22-H05-sc0414 V_(L) of humanized scFv of clone 16-22-H05-sc02 15 Humanized scFv ofclone 16-22-H05-sc02 16 CDR L1 of clone 16-12-B11 17 CDR L1 of clone16-13-C08 18 CDR L1 of clone 16-13-E05 19 CDR L1 of clone 16-16-B08 20CDR L1 of clone 16-16-H10 21 CDR L1 of clone 17-13-G07 22 CDR L1 ofclone 17-20-E06 23 CDR L2 of clone 16-12-B11, clone 16-16-B08 and clone17-13-G07 24 CDR L2 of clone 16-13-C08 25 CDR L2 of clone 16-13-E05 26CDR L2 of clone 17-20-E06 27 CDR L3 of clone 16-12-B11 28 CDR L3 ofclone 16-13-C08 29 CDR L3 of clone 16-13-E05 30 CDR L3 of clone16-16-B08 31 CDR L3 of clone 17-13-G07 32 CDR L3 of clone 17-20-E06 33CDR H1 of clone 16-12-B11 and clone 16-13-E05 34 CDR H1 of clone16-13-C08 35 CDR H1 of clone 17-13-G07 36 CDR H1 of clone 17-20-E06 37CDR H2 of clone 16-12-B11 and clone 17-13-G07 38 CDR H2 of clone16-13-C08 39 CDR H2 of clone 16-13-E05 40 CDR H2 of clone 16-16-B08 andclone 17-20-E06 41 CDR H2 of clone 16-16-H10 42 CDR H3 of clone16-12-B11 43 CDR H3 of clone 16-13-C08 44 CDR H3 of clone 16-13-E05 45CDR H3 of clone 16-16-B08 46 CDR H3 of clone 16-16-H10 47 CDR H3 ofclone 17-13-G07 48 CDR H3 of clone 17-20-E06 49 Linker sequence in scFv50 Linker sequence in diabody 51 Humanized diabody of clone 16-22-H05 52Light chain of humanized IgG of clone 16-22-H05 53 Heavy chain ofhumanized IgG of clone 16-22-H05 54 V_(L) of humanized scFv of clone16-22-H05-sc04 55 Humanized scFv of clone 16-22-H05-sc04 56 Vκ1consensus sequence of framework I (Kabat positions 1-23) 57 Vκ1consensus sequence of framework II (Kabat positions 35-49) 58 Vκ1consensus sequence of framework III (Kabat positions 57-88) 59 Vλgermline-based sequence of framework IV (see Table 16) 60 Vλgermline-based sequence of framework IV (see Table 16) 61 Vλgermline-based sequence of framework IV (see Table 16) 62 Vλgermline-based sequence of framework IV (see Table 16)

EXAMPLES Example 1: Generation of Rabbit Antibodies Directed AgainstHuman TNFα

1. Results

1.1 Immunization

Rabbits have been immunized with purified recombinant human TNFα(Peprotech, Cat. No. 300-01A). During the course of the immunization,the strength of the humoral immune response against the antigen wasqualitatively assessed by determining the maximal dilution (titer) forthe serum of each rabbit that still produced detectable binding of thepolyclonal serum antibodies to the antigen. Serum antibody titersagainst immobilized recombinant human TNFα were assessed using anenzyme-linked immunosorbent assay (ELISA, see 2.2.1). All three rabbitsshowed very high titers with a 10×10⁶-fold dilution of the serum stillresulting in a positive signal (at least 3-fold higher than the signalobtained with serum from a naïve unrelated animal which was used asbackground control) in the ELISA. In addition, the ability of differentrabbit sera to inhibit the biological activity of TNFα was assessedusing a mouse L929 cell-based assay (see 2.2.3). All three serainhibited TNFα-induced apoptosis of mouse L929 fibroblasts. Rabbit #3showed the strongest neutralizing activity with 50% inhibition (IC₅₀)reached at a serum dilution of 1:155′000. Compared to rabbit #3, rabbit#2 and rabbit #1 showed approximately 3 and 21-fold lower activity,reaching 50% inhibition at a serum dilution of 1:55′500 and 1:7′210,respectively.

Lymphocytes isolated from spleens of all three animals were chosen forthe subsequent hit identification procedures. The animals wereprioritized based on the potency to inhibit the biological activity ofTNFα in the L929 assay. Therefore, the highest number of hits that werecultivated originated from rabbit #3, and the lowest number of hits wasderived from rabbit #1.

1.2 Hit Identification

1.2.1 Hit Sorting

Prior to the hit identification procedure, a flow-cytometry-basedsorting procedure was developed that specifically detects and allows forthe isolation of high-affinity TNFα binding B-cells (see 2.1).

A total of 33×10⁶ lymphocytes (corresponding to 1.5% of totallymphocytes isolated) derived from all three rabbits were characterizedin two independent sorting campaigns. Out of the 33×10⁶ cells analyzedin total 3452 B-cells expressing TNFα-specific antibodies (IgG) wereisolated. The numbers of lymphocytes cloned were different for the threerabbits, as more cells were isolated from those rabbits whose serashowed strong inhibition of TNFα in the L929 assay. Of the isolatedB-cells, 792 clones were derived from rabbit #1, 1144 clones from rabbit#2 and 1408 clones from rabbit #3. For 108 clones the respective rabbitorigin is not known, because they are derived from a mixture of residuallymphocytes from all 3 rabbits to allow optimal recovery of small amountof lymphocytes from the vials.

1.2.2 Hit Screening

The results obtained during the screening phase are based on assaysperformed with non-purified antibodies from culture supernatants ofantibody secreting cells (ASC), as the scale of the high-throughputculture does not allow for purification of the individual rabbitantibodies. Such supernatants were used to rank large numbers ofantibodies relative to each other, however not to provide absolutevalues (e.g. for inhibition of biological activity of TNFα), except forbinding affinity. ASC supernatants were screened in a high-throughputELISA for binding to recombinant human TNFα. TNFα-binding supernatantswere further characterized for binding to Cynomolgus monkey TNFα byELISA, binding kinetics and for their potential to neutralize thebiological activity of human TNFα in the L929 assay. With the exceptionof binding kinetics, the reporting values of the high-throughputscreenings should be interpreted as “yes” or “no” answers, which arebased on single-point measurements (no dose-response). Affinity toCynomolgus monkey and mouse TNFα was analyzed for all the 102 clonesthat were selected for amplification and sequencing of the antibodyheavy and light chain variable domains.

1.2.2.1 Binding to Human TNFα

The aim of the primary screening is to identify ASC clones that produceantibodies specific for human TNFα. For this purpose, cell culturesupernatants of 3452 ASC clones were analysed for the presence ofantibodies to human TNFα by ELISA (see 2.2.1). The ELISA method usedassesses the “quantity” of antibodies of the IgG subtype bound torecombinant human TNFα, gives however no information about the affinityor the concentration of the antibodies. In this assay, supernatants from894 ASC clones produced a signal that was clearly above background. Thehit rate in screening was similar for rabbit #1 and rabbit #2 with 153hits out of 792 (19.3%) identified from rabbit #1 and 225 hits out of1144 identified from rabbit #2 (19.7%). Rabbit #3 showed a significantlyhigher hit rate of 34.4% resulting in the identification of 484 hits outof 1408. All 894 hits identified in this primary screening, weresubjected to the measurement of binding kinetics by SPR (secondaryscreening).

1.2.2.2 TNFα Binding Kinetics

The aim of the secondary screening is to obtain quantitative informationon the quality of target binding for each hit from the primary screeningby surface plasmon resonance (SPR, see 2.2.2). In contrast to the ELISAused during the primary screening, this method assesses the kinetics oftarget binding as a function of time. This allows determination of therate constants for association (k_(a)) and dissociation (k_(d)) of theantibody from its target. The ratio k_(d)/k_(a) provides the equilibriumdissociation constant (K_(D)), which reflects the affinity of anantibody to its target. Of the 894 hits identified in the primaryscreening, binding affinities to human TNFα could be determined for 839monoclonal rabbit antibodies. For the remaining 55 antibodies affinitycould not be measured because the antibody concentration in the ASCsupernatant was below the detection limit of the SPR instrument in therespective setup. The 839 anti-TNFα antibodies that could be measuredshowed dissociation constants (K_(D)) ranging from below 1.36×10⁻¹³ M to1.14×10⁻⁸ M. 69% of all antibodies analyzed had a K_(D) below 0.5 nM.

The median K_(D)s of 2.21×10⁻¹⁰ M and 2.09×10⁻¹⁰ M for screening hitsidentified from rabbits #2 and #3 were similar while rabbit #1 showedabout 2-fold higher values with a median K_(D) of 4.65×10⁻¹⁰ M. Whenconsidering only neutralizing screening hits, the affinity distributionswere similar for all three animals with lower values for the medianK_(D) (median K_(D)s between 1.4×10⁻¹⁰ M and 1.27×10⁻¹⁰ M). Affinitiesbelow 0.041 nM, 0.029 nM and 0.026 nM were measured for 5% of screeninghits for rabbits #1, #2 and #3, respectively. For 2% of supernatants,affinities were even in the low picomolar range (below 6.2 pM, 7.9 pMand 11 pM). The excellent yield of high-affinity antibodies resultingfrom the secondary screening provides a broad basis for the selection ofthe most appropriate antibodies for humanization and reformatting.

1.2.2.3 Potency

For the assessment of potency, a cell-based assay (L929 assay) has beendeveloped (see 2.2.3). 506 out of the 894 selected antibodies (56.6%),inhibited TNFα-induced apoptosis in the L929 assay by more than 50%. Inline with results obtained during titer analysis, the highest percentageof neutralizing hits was derived from rabbit #3 with a hit rate of62.8%, followed by rabbit #2 with a hit rate of 56.4% and rabbit #1 withthe lowest hit rate of 39.9%. Affinities of these neutralizingantibodies ranged between 1.36×10⁻¹³ to 1.19×10⁻⁹M.

1.2.2.4 Species Cross-Reactivity (Cynomolgus Monkey)

All 894 hits identified in the primary screening, were analyzed forspecies cross-reactivity to Cynomolgus monkey TNFα by ELISA (see 2.2.1).The aim of this additional screening was to allow selection of ASCclones that are known to cross-react with Cynomolgus monkey TNFα. TheELISA method used assesses the “quantity” of antibodies of the IgGsubtype bound to recombinant Cynomolgus monkey TNFα, gives however noinformation about the affinity or the concentration of the antibodies.Supernatants from 414 (46%) ASC clones produced a clear signal (opticaldensity (OD)≥1). The percentage of hits cross-reactive to Cynomolgusmonkey TNFα was similar for rabbit #1 and rabbit #3 with 81 hits out of153 (52.9%) identified from rabbit #1 and 236 hits out of 484 identifiedfrom rabbit #3 (48.8%). With 37.8%, rabbit #2 showed a slightly lowerpercentage of cross-reactive hits resulting in the identification of 82hits out of 225.

1.2.2.5 Selection of Clones for RT-PCR

As a prerequisite for hit confirmation, gene sequence analysis andsubsequent humanization of the rabbit antibodies, the geneticinformation encoding the rabbit antibody variable domain needs to beretrieved. This was done by reverse transcription (RT) of the respectivemessenger RNA into the complementary DNA (cDNA), followed byamplification of the double stranded DNA by the polymerase chainreaction (PCR). The selection of ASC clones subjected to RT-PCR wasprimarily based on affinity and neutralizing activity. As additionalcriterion cross-reactivity to Cynomolgus monkey TNFα was considered. Intotal 102 ASC clones were selected for gene cloning by RT-PCR. First,the 93 best ranking ASC (in terms of affinity) with a K_(D) below 80 pM,that inhibited the biological activity of TNFα in the L929 assay by morethan 50% and that showed significant binding to Cynomolgus monkey TNFαwere selected. Additionally, all the 9 best ranking ASC clones withK_(D) below 20 pM that neutralized TNFα activity by more than 50% butdid not bind to Cynomolgus monkey TNFα nevertheless were chosen as well.In total, 12, 13 and 66 ASC clones were successfully amplified andsequenced from rabbits #1, #2 and #3, respectively.

1.2.2.6 Identification of Related Clones with Desired Properties

In order to characterize the genetic diversity of the panel of isolatedASC clones the sequences of the complementary determining regions (CDRs)were extracted and subjected to a multiple sequence alignment thusallowing sequence clustering in a phylogenetic tree.

While this analysis on one hand allows the selection of a diverse set ofclonal sequences to be carried forward into humanization andre-formatting experiments it also identifies homologous clusters ofclonal sequences that appeared to share a common parental B-cell clonein the rabbit. The hallmark of these sequence clusters are high sequencehomology in the CDRs and a consistent pattern of pharmacodynamicproperties. Both of these features are summarized for a cluster of eightclones in Tables 2 and 3. Despite the functional conservation of thissequence cluster the consensus sequence in Table 3 reveals that acertain variability of the CDRs is tolerated, while still resulting inthe desired pharmacodynamic profile.

TABLE 2 Pharmacodynamic properties of monoclonal antibodies in ASCsupernatants.. Affinity to human TNFα Affinity to Cynomolgus TNFα L929ASC SN k_(a) K_(d) K_(D) k_(a) K_(d) K_(D) assay Clone ID (M⁻¹s⁻¹) (s⁻¹)(M) (M⁻¹s⁻¹) (s⁻¹) (M) % inh. 16-12-B11 3.22E+06 1.87E−04 5.82E−112.15E+06 1.37E−03 6.34E−10 71 16-13-C08 1.77E+06 9.41E−05 5.33E−111.80E+06 1.77E−04 9.82E−11 93 16-13-E05 2.78E+06 8.27E−05 2.97E−112.53E+06 2.99E−04 1.18E−10 94 16-16-B08 2.27E+06 4.53E−05 1.99E−112.35E+06 1.52E−04 6.45E−11 73 16-16-H10 2.03E+06 1.59E−04 7.85E−112.20E+06 4.61E−04 2.10E−10 70 16-22-H05 1.90E+06 7.26E−05 3.82E−112.17E+06 6.97E−05 3.21E−11 67 17-13-G07 7.87E+05 1.37E−06 1.73E−127.80E+05 7.34E−05 9.41E−11 109 17-20-E06 1.19E+06 3.59E−06 3.03E−121.45E+06 3.27E−05 2.26E−11 101

TABLE 3 The following sequence data regarding theCDRs were obtained for the above clones: SEQ ID CDR clone Sequence* NO:CDR L1 16-22-H05 QASQSIFSGLA 7 16-12-B11 QASQSISNYLA 16 16-13-C08QASQSISTALA 17 16-13-E05 QASQSIGRNLA 18 16-16-B08 QASQSISNSLA 1916-16-H10 QASQSIYSGLA 20 17-13-G07 QASQSIGSNLA 21 17-20-E06 QASQSISSSLA22 QASQSIXXXLA 1 CDR L2 16-22-H05 GASKLAS 8 16-12-B11 RASTLAS 2316-13-C08 RASTLES 24 16-13-E05 QASKLAS 25 16-16-B08 RASTLAS 23 16-16-H10GASKLAS 8 17-13-G07 RASTLAS 23 17-20-E06 RASKLAS 26 XASXLXS 2 CDR L316-22-H05 QSYYYSSSSSDGSYA 9 16-12-B11 QSYYYSSSSSDGFFA 27 16-13-C08QSYYYSSSSSDGSFA 28 16-13-E05 QSYYYSSSNSDGSLA 29 16-16-B08QSYYYSSISSDGSYA 30 16-16-H10 QSYYYSSSSSDGSYA 9 17-13-G07 QSYYYSSSSSDGSVA31 17-20-E06 QSYYYTSSTSDGSYA 32 QSYYYXSXXSDGXXA 3 CDR H1 16-22-H05GIDFNNYGIG 10 16-12-B11 GIDFSNYGIC 33 16-13-C08 GIDFSNYGIS 34 16-13-E05GIDFSNYGIC 33 16-16-B08 GIDFNNYGIG 10 16-16-H10 GIDFNNYGIG 10 17-13-G07GIDFSTYGIS 35 17-20-E06 GIDFSNYGIG 36 GIDFXXYGIX 4 CDR H2 16-22-H05YIYPGFAITNFANSVKG 11 16-12-B11 YIYPGFGITNYAMSVKG 37 16-13-C08YIYPGFGIRNYAHSVKG 38 16-13-E05 YIYPGFGIRNYANSLKG 39 16-16-B08YIYPGFAIRNYAMSVKG 40 16-16-H10 YIYPGFGITNFANSVKG 41 17-13-G07YIYPGFGITNYAMSVKG 37 17-20-E06 YIYPGFAIRNYANSVKG 40 YIYPGFXIXNXAXSXKG 5CDR H3 16-22-H05 DPVYATSSGYFDL 12 16-12-B11 DPIYASSSGYLDL 42 16-13-C08DPVYSSDWGYFNL 43 16-13-E05 DPVYASSSGYLDL 44 16-16-B08 DPLYATSSGYFDL 4516-16-H10 DPVYASSSGYFDL 46 17-13-G07 DPVYASSSAYYNL 47 17-20-E06DPLYSTSSGYFNL 48 DPXYXXXXXYXXL 6 *Amino acids designated “X” have themeaning as defined in the accompanying sequence listing.

1.2.2.7 Cross-Reactivity to Cynomolgus Monkey TNFα (by SPR)

Because of the high number of high affinity hits that potentlyneutralized TNFα, species cross-reactivity was assessed for allmonoclonal rabbit antibodies that were subjected to RT-PCR in order tofacilitate the selection of ASC clones for Hit confirmation. Affinitiesto Cynomolgus monkey TNFα were determined by SPR measurements similarlyas described above (see also 2.2.2). The affinities of the 93 testedantibodies for Cynomolgus monkey TNFα ranged from 9.6×10⁻¹² to 2.1×10⁻⁹M. 38 of the 93 cross-reactive antibodies bound human and CynomolgusTNFα with similar affinity (less than two-fold difference in K_(D)).Moreover, the difference in affinity between human and Cynomolgus wasless than 20-fold for 79 of the 93 cross-reactive antibodies and lessthan 10-fold for 62 of them, which makes them acceptable for thepreclinical development in the Cynomolgus monkey.

2. Methods

2.1 Sorting Assay

Flow-cytometry based sorting procedure for the isolation ofantigen-specific B-cells from rabbit lymphatic tissue was performed asoutlined by Lalor et al (Eur J Immunol. 1992; 22.3001-2011)

2.2 Screening Assays

2.2.1 TNFα Binding ELISA (human and Cynomolgus monkey TNFα) Recombinanthuman TNFα (Peprotech, Cat. No. 300-01) was coated on a 96 wellmicrotiter ELISA plate. Binding of rabbit antibodies in the ASC culturesupernatants to the immobilized TNFα was detected by a secondaryHRP-labelled anti-rabbit IgG (JacksonImmuno Research, Cat. No.111-035-046). TMB substrate (3,3′,5,5′-tetramethylbenzidine, KPL, Cat.No. 53-00-00) was added and the colour reaction was stopped by theaddition of H₂SO₄. Plates were read using a microtiter plate reader(Infinity reader M200 Pro, Tecan) at a wavelength of 450 nm.

Assay performance during the screening campaigns was monitored by acommercially available positive control anti-TNFα rabbit polyclonalantibody (AbD Serotec, Cat. No. 9295-0174). For this purpose thepositive control antibody was tested at 100 and 250 ng/ml in duplicateon each screening plate. Robustness and precision of the response of thepositive control was monitored for each plate. At the final assayconditions, the signal-to-background ratio was between 30 to 40 for thepositive control at 250 ng/ml and coefficient of variation (CV) of thepositive control were below 10%. A signal with an optical density of100% relative to the 250 ng/ml positive control was considered as aprimary screening hit.

For serum titer determinations, the same ELISA setup was used asdescribed above. A serum dilution was considered positive when thebinding signal of the immune serum was at least 3-fold higher comparedto the signal of a naïve unrelated animal.

Species cross-reactivity to Cynomolgus monkey was determined using asimilar ELISA as described above. Recombinant Cynomolgus monkey TNFα(Sino Biological, Cat. No. 90018-CNAE) was coated on 96 well microtiterELISA plates. Binding of rabbit antibodies in the ASC culturesupernatants to the immobilized Cynomolgus monkey TNFα was detected bythe HRP-labelled secondary antibody as specified above. Immune serumfrom rabbit #2 was used as positive control at a dilution of 1:80′000and 1:320′000. Robustness and precision of the response of the positivecontrol was monitored for each plate. At the final assay conditions, thesignal-to-background ratio was between 20 to 30 for the positive controlat a dilution of 1:80′000 and CVs of the positive control were below10%.

2.2.2 Binding Kinetics to TNFα by SPR (Human and Cynomolgus Monkey)

Binding affinities of antibodies towards human TNFα were measured bysurface plasmon resonance (SPR) using a MASS-1 SPR instrument (SierraSensors). Performance of the instrument was qualified by means ofstandard reference solutions as well as by analysis of a referenceantibody-antigen interaction such as infliximab-TNFα interaction.

For affinity screening, an antibody specific for the Fc region of rabbitIgGs (Bethyl Laboratories, Cat. No. A120-111A) was immobilized on asensor chip (SPR-2 Affinity Sensor, High Capacity Amine, Sierra Sensors)using a standard amine-coupling procedure. Rabbit monoclonal antibodiesin ASC supernatants were captured by the immobilized anti-rabbit IgGantibody. After capturing of the monoclonal antibodies, human TNFα(Peprotech, Cat. No. 300-01) was injected into the flow cells for 3 minat a concentration of 90 nM, and dissociation of the protein from theIgG captured on the sensor chip was allowed to proceed for 5 min. Aftereach injection cycle, surfaces were regenerated with two injections of10 mM Glycine-HCl. The apparent dissociation (k_(d)) and association(k_(a)) rate constants and the apparent dissociation equilibriumconstant (K_(D)) were calculated with the MASS-1 analysis software(Analyzer, Sierra Sensors) using one-to-one Langmuir binding model andquality of the fits was monitored based on relative Chi² (Chi²normalized to the extrapolated maximal binding level of the analyte),which is a measure for the quality of the curve fitting. For most of theHits the relative Chi² value was below 15%. Results were deemed valid ifthe response units (RU) for ligand binding were at least 2% of the RUsfor antibody capturing. Samples with RUs for ligand binding with lessthan 2% of the RUs for antibody capturing were considered to show nospecific binding of TNFα to the captured antibody.

Species cross-reactivity to Cynomolgus monkey TNFα (Sino Biological,Cat. No. 90018-CNAE) was measured using the same assay setup and TNFαconcentrations and applying the same quality measures. The relative Chi²was below 15% for most of the ASC supernatants analyzed.

2.2.3 TNFα-Induced Apoptosis in L929 Fibroblasts

The ability of rabbit IgGs from ASC culture supernatants to neutralizethe biological activity of recombinant human TNFα was assessed usingmouse L929 fibroblasts (ATCC/LGC Standards, Cat. No. CCL-1). L929 cellswere sensitized to TNFα-induced apoptosis by addition of 1 μg/mlactinomycin D. Cells were cultured in 96-well flat-bottom microtiterplates in the presence of 50% ASC culture supernatant and 100 pM (5.2ng/ml) human TNFα (Peprotech, Cat. No. 300-01) for 24 h. Compared topurified antibodies, higher concentrations of TNFα have to be used inthe presence of ASC supernatants for hit screening. Survival of thecells was determined by a colorimetric assay using the WST-8(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt) cell proliferation reagent (Sigma Aldrich, Cat. No.96992). WST-8 is reduced by cellular dehydrogenases to an orangeformazan product. The amount of formazan produced is directlyproportional to the number of living cells. Data were analyzed using afour-parameter logistic curve fit using the Softmax Data AnalysisSoftware (Molecular Devices), and the concentration of infliximabrequired to neutralize TNFα-induced apoptosis by 50% (IC₅₀) wascalculated at a concentration of 36.2 ng/ml. Therefore, the estimatedlower limit of detection for this assay is between 30 to 40 ng/ml. Thisvalue is only a rough estimate for the detection limit, since thepotential to block TNFα is not only dependent on the concentration ofthe monoclonal antibody but also on affinity of the antibody to thetarget. However, the sensitivity of the assay is sufficient forscreening of ASC supernatants since IgG concentrations in most ASCsupernatants are above a concentration of 40 ng/ml. Supernatantsresulting in 50% neutralization of TNFα-induced apoptosis wereconsidered positive.

To assure robust assay performance during the screening campaigns thepositive control antibody infliximab was tested at 115 ng/ml (0.8 nM)and at 58 ng/ml (0.4 nM) in duplicates on each screening plate. Percentinhibition and precision of the response for the positive control wasmonitored for each screening plate. The acceptance criteria for eachplate were set as follows: at least 60% inhibition with the positivecontrol antibody at a concentration of 115 ng/ml with a coefficient ofvariation (CV) below 20%.

Example 2: Humanization and Generation of scFv

1. Results

1.1 Hit Confirmation & Selection of Hits for Humanization

73 unique sets of parental rabbit light and heavy chain variable domainswere retrieved during hit screening and analyzed by sequence alignment.Based on the screening assay results and the sequence homology of theindividual rabbit IgG clones, 30 candidates were selected for hitconfirmation. 29 monoclonal antibodies were manufactured and the bestperforming clones in terms of affinity and potency were selected for thehumanization and lead candidate generation. The criteria for theselection of clones were i) neutralization of human TNFα in L929 assay,ii) high affinity to human TNFα, iii) cross-reactivity to Cynomolgus andRhesus monkey TNFα, and iv) sequence diversity. One clone (16-22-H05)has been selected for humanization—one of the best ranking IgGs in termsof potency to neutralize human TNFα in the L929 assay. With respect tobinding strength, the best possible affinity is desired since a certainloss of affinity needs to be anticipated as a result of humanization andreformatting into the scFv format.

The data for IgG clone No. 16-22-H05 are summarized in Table 4.

TABLE 4 In vitro binding and activity properties of purified monoclonalantibody (16-22-H05) Neutraliza- Blocking Blocking tion of of TNF- ofTNF- Affinity to human TNF Affinity to cyno TNF Affinity to rhesus TNFTNF in TNFR1 TNFR2 k_(a) K_(d) KD k_(a) k_(d) KD k_(a) k_(d) KD L929assay interaction interaction (M⁻¹s⁻¹) (s⁻¹) (M) (M⁻¹s⁻¹) (s⁻¹) (M)(M⁻¹s⁻¹) (s⁻¹) (M) rel. IC₅₀* rel. IC₅₀* rel. IC₅₀* 4.04E+06 1.31E−043.25E−11 3.50E+06 4.89E−06 1.40E−12 2.01E+06 5.31E−04 2.65E−10 9.95 1.020.88 *IC_(50, Infliximab)/IC_(50, Test Sample)

1.2 Generation and Selection of Humanized scFv Fragments

The sequences encoding the complementarity determining regions (CDRs)were transferred in silico by CDR-loop grafting onto a human variabledomain scaffold sequence as described in WO 2014/206561. In addition asecond construct was generated per rabbit clone, which transferredadditional amino acids from the donor sequence at positions withstructural relevance for the immunoglobulin domains and CDR positioning.An artificial gene (with an optimized codon usage for bacterialexpression) encoding the respective humanized single-chain antibody Fv(scFv) was synthesized (from the corresponding variable light and heavychains). The polypeptide was then produced and subsequentlycharacterized using similar assays as described during the hitconfirmation.

1.2.1 Humanization and Manufacture of Humanized scFv (APIs)

The humanization of the selected clone comprised the transfer of therabbit CDRs onto a scFv acceptor framework of the Vκ1/VH3 type asdescribed in WO 2014/206561. In this process, which is schematicallyshown in FIG. 1, the amino acid sequence of the six CDR regions wasidentified on the donor sequence (rabbit mAb) and grafted into theacceptor scaffold sequence, resulting in the constructs termed “CDRgraft”.

In addition, a second graft was designed, which included additionalamino acids modifications from the rabbit donor on the positions L15,L22, L48, L57, L74, L87, L88, L90, L92, L95, L97, L99 and H24, H25, H56,H82, H84, H89, H108 (AHo numbering), which have been described topotentially influence CDR positioning and thus antigen binding (Borraset al. JBC. 2010; 285:9054-9066). These humanized construct is termed“structural (STR) graft”. In case the comparison of the characterizationdata for these two initial constructs revealed a significant advantageof the STR construct additional variants were designed that combined theCDR grafted VL with STR grafted VH. This combination has been proven tobe often sufficient to retain the activity of the STR graft (Borras etal., 2010, JBC, 285:9054-9066) and would generally be preferred as fewernon-human alterations in the human acceptor scaffold reduce the risk forimpaired stability and also the potential for immunogenicity.

Once the in-silico construct design described in the previous sectionwas completed the corresponding genes were synthesized and bacterialexpression vectors were constructed. The sequence of the expressionconstructs was confirmed on the level of the DNA and the constructs weremanufactured according to generic expression and purification protocols.

The heterologous expression of the proteins was performed in E. coli asinsoluble inclusion bodies. The expression culture was inoculated withan exponentially growing starting culture. The cultivation was performedin shake flasks in an orbital shaker using commercially available richmedia. The cells were grown to a defined OD₆₀₀ of 2 and induced byovernight expression with 1 mM Isopropyl β-D-1-thiogalactopyranoside(IPTG). At the end of fermentation the cells were harvested bycentrifugation and homogenized by sonication. At this point theexpression level of the different constructs was determined by SDS-PAGEanalysis of the cell lysate. The inclusion bodies were isolated from thehomogenized cell pellet by a centrifugation protocol that includedseveral washing steps to remove cell debris and other host cellimpurities. The purified inclusion bodies were solubilized in adenaturing buffer (100 mM Tris/HCl pH 8.0, 6 M Gdn-HCl, 2 mM EDTA) andthe scFvs were refolded by a scalable refolding protocol that generatedmilligram amounts of natively folded, monomeric scFv. A standardizedprotocol was employed to purify the scFvs, which included the followingsteps. The product after refolding was captured by an affinitychromatography employing Capto L agarose (GE Healthcare) to yield thepurified scFvs. Lead candidates that met the affinity and potencycriteria in initial testing were further purified by a polishingsize-exclusion chromatography using a HiLoad Superdex75 column (GEHealthcare). Subsequent to the purification protocol the proteins wereformulated in a buffered saline solution and characterized by thevarious biophysical, protein interaction and biological methods, asdescribed in the following. The producibility of the differentconstructs was compared by determining the final yield of purifiedprotein for the batch and normalizing this value to 1 L of refoldingvolume.

1.2.2 Biophysical Characterization of Humanized scFv

The biophysical characterization of the scFv with respect to stabilityand producibility were compiled in Table 5. The producibility andstability of the scFv construct was characterized by the differentreporting points as discussed in the subsequent sections.

The scFv was investigated as to certain criteria, as explained in thefollowing.

The producibility criterion shall ensure that the selected scFv entitycan be expressed, refolded and purified in sufficient amounts to supportlater development of the lead molecule. The defined criteria were theexpression yield of scFv per liter of fermentation broth, as assessed bySDS-PAGE, and the purification yield achieved in the generic lab-scaleprocess, as assessed by measurement of the amount of purified protein byUV spectrometry, calculated back to 1 liter of refolding solution.

The criteria for stability were intended to assess the aggregationpropensity during the manufacturing process of the molecules and theirstructural integrity during storage and further handling. The monomercontent determined by SE-HPLC allows assessing the colloidal stabilityof molecules during the purification process (2.2.3). In a subsequentstability study the monomer content was tested over a duration of 4weeks at 1 and 10 mg/mL and storage at 4, −20 and <−65° C. In addition,the colloidal stability of the proteins was tested after 5 freezing andthawing cycles. As an additional stability indicating parameter, themidpoint of thermal unfolding was determined by differential scanningfluorimetry (DSF) (2.2.4) to provide a read-out for the conformationalstability of the lead candidates.

TABLE 5 Summary of the biophysical characterization data for thehumanized scFvs. Stability Producibility Freeze/ Purification Tm Storage[Δ %] Thaw Purity Expression Yield Clone ID Construct [° C.] −65° C.−25° C. 4° C. [Δ %] [%] [g/L] [mg/L] 16-22-H05-sc01 16-22-H05 CDR 74.2nd nd nd nd 98.0 nd 19.1 16-22-H05-sc02 STR 71.3 0.0 0.0 0.2 0.0 99.90.27 29.8 16-22-H05-sc04 CDR/STR 72.6 0.2 0.1 0.4 0.0 100.0 0.26 24.1

1.2.2.1 Producibility Assessment

The lead candidate scFv molecules were expressed by shake flaskfermentation in batch mode and purified by a generic lab-scale processto yield the protein samples for further characterization. During thisprocess some key performance parameters were monitored to compare thecandidate molecules and to identify potentially difficult to developconstructs.

The expression titer was determined on the level of the crude E. colilysate after the harvest of the cells by centrifugation. During theharvest a small loss of cells is anticipated, however, this factor waschosen to be neglected for the calculation of the expression yield infavor of a more conservative estimation of the productivity. For thequantification of the scFv product in the lysate coomassie stainedreducing SDS-PAGE (2.2.1) was chosen due to the high specificity of themethod that allows discriminating the product from the host cellproteins in the sample.

A second criterion to assess the producibility is the purification yieldof scFv calculated per liter of refolding solution. This parameteraddresses the potential bottleneck in the anticipated manufacturingprocess that includes a protein refolding step. Since the efficiency ofthe refolding procedure has proven to be limiting in comparablemanufacturing processes it has been decided to compare the performanceof the different constructs with respect to the producibility normalizedto a defined refolding volume. For the calculation of the yield thefinal protein sample from each batch was quantified by UV absorbance(2.2.2) and divided by the actual refolding volume of the respectivepurification (Table 6).

TABLE 6 Summary of producibility data for two humanized scFvs. Theexpression titer was determined by quantitative SDS-PAGE on lysates ofend-of-production cells. The batch yield was determined by UV absorbancemeasurement of the final purification pool. The purification yield iscalculated as the purified scFv per liter of refolding volume.Producibility Expression Batch Refolding Purification Titer Yield VolumeYield Construct ID [g/L] [mg] [L] [mg/L] 16-22-H0S-sc02 0.27 14.3 0.4829.8 16-22-H05-sc04 0.26 11.9 0.49 24.1

1.2.2.2 Stability Assessment

The assessment of the conformational stability, monodispersity andstructural integrity of the scFv constructs is an integral component forthe ranking of the different molecules with respect to thedevelopability. A prerequisite for the meaningful comparison of thedifferent constructs is the preparation of purified molecules of similarquality. The criterion “monomer purity” determined by SE-HPLC isintended to ensure compatible quality of the different test substances.In addition to the SE-HPLC analysis, SDS-PAGE for the determination ofprotein purity and identity was performed to confirm comparable qualityof the tested preparations.

The SE-HPLC results of the two scFvs reveal that all preparations couldbe purified to a monomer content of ≥99% (FIG. 2).

The thermal unfolding behavior of the lead candidates was tested bydifferential scanning fluorimetry (DSF) to allow ranking of themolecules with respect to their expected conformational stability. Anormalized plot of the fluorescence raw data is shown in FIG. 3, whichdepicts duplicate measurements of each sample. A cooperative unfoldingbehavior was observed. The two molecules 16-22-H05-sc02 and16-22-H05-sc04 showed a T_(m) of 71.3 and 72.6° C., respectively.

In a second arm of the stability assessment the monodispersity of themolecules was monitored over the duration of 4 weeks at differenttemperatures. The results for the stability study and the resultingmonomer contents are shown in FIG. 4. Both molecules (16-22-H05-sc02 and16-22-H05-sc04) start at a monomer content exceeding the minimum of 95%monomer and lose less than 5% of monomer with respect to the respectivestarting value at a concentration of 10 mg/ml. In the frozen state at−20° C. and <−65° C. the samples only showed minimal differences overtime. At the most stringent condition (4° C.) the molecule16-22-H05-sc02 lost as little as 0.2% of monomer during the 4 weeks. Inaddition a stress stability study was conducted at a temperature of 37°C. and a scFv concentration of 10 mg/ml for up to 4 weeks. At thiscondition a more stringent discrimination of the propensity foraggregation of the different constructs is expected. The resulting datasummarized in FIG. 6 revealed a monomer loss of 15% after 28 days. BothscFv demonstrated good monomer stability at stress conditions.Chromatograms of the stability study at 4° C. are provided in FIG. 5,where the sample at day 0 and after 28 days at 4° C. is shown. In thischromatogram overlay also the results of the freeze/thaw stability isshown. For this part of the study the samples were repeatedly frozen andthawed for a total of 5 cycles. The resulting quantification of themonomer content by analytical SE-HPLC did not reveal any changes in thetwo samples (Table 5).

A SDS-PAGE analysis was performed for the two scFvs to generatesupportive data for the quantification by UV absorbance, confirming thepurity of the sample preparation and thereby conferring specificity forthe content quantification. In another aspect of this analysis theSDS-PAGE results revealed the absence of protein degradation during thestability study (28 days at 4° C. and a concentration of 10 mg/mlcompared to sample from day 0 stored at <−65° C.), which is an importantcharacteristic from a developability perspective.

It is important to note that the different studies performed within thescope of this assessment address distinct mechanistic aspects of proteinstability. The determination of the thermal unfolding temperature of aprotein will give complementary results to the measurement of themonodispersity by SE-HPLC upon storage at elevated temperature. Whileboth methods are designed to give an estimation of the potential productshelf live and stability the mechanisms addressed are profoundlydifferent. The midpoint of transition (Tm) assessed by thermal unfoldingis a qualitative measure for protein domain stability (does not allowfor thermodynamic determination of ΔG). Highly stable protein domains(high Tm) are less likely to spontaneously unfold at ambient temperatureand thus less prone to irreversible aggregation/precipitation driven byunfolded domain interactions. High domain stability indicates densepackaging of amino acid residues, which also correlates with resistancetowards protease cleavage. The SE-HPLC assessment on the other handquantitatively determines the content of the monomeric fraction as wellas of soluble oligomers/aggregates. Such soluble oligomers areoftentimes reversible and relatively loose associations driven byelectrostatic or hydrophobic interactions between correctly foldedproteins. There is some correlation between Tm as assessed by thermalunfolding and the propensity for oligomer/aggregate formation asassessed by SE-HPLC particularly for proteins with “border line”stability. Beyond a certain threshold Tm of approximately 60° C.antibody variable domains are generally sufficiently stable to beresistant toward aggregation/precipitation and proteolytic degradationdue to partial domain unfolding at ambient temperature. Oligomerizationdriven by hydrophobic and/or electrostatic interactions of surfaceresidues may, however, still occur. Importantly, in an accelerated(stress) stability study at elevated temperature (e.g. 37° C.) thevarious mechanisms of oligomer formation and precipitation may occursimultaneously.

1.2.3 Characterization of In Vitro Binding and Activity of HumanizedscFvs

In the following the humanized scFvs were characterized in vitro fortheir target binding properties and potencies. Binding kinetics (ka, kdand KD) to human TNFα and potency to neutralize TNFα-induced apoptosisof L929 fibroblasts was analyzed. Additionally, the potency to inhibitCynomolgus monkey (Macaca fascicularis) and Rhesus monkey (Macacamulatta) TNFα induced apoptosis as well as the potency to inhibit theinteraction between human TNFα and TNFRI/TNFRII by ELISA and targetselectivity for binding to TNFα over TNFβ was determined.

For the understanding of the results below it is important to note thatboth, the transfer of the rabbit CDRs onto a human variable domainscaffold as well as the change in the format from the full-size IgG tothe scFv fragment may impact on pharmacological properties. For example,a certain loss of affinity is usually associated with humanization.Further, due to the smaller size of the scFv compared to the IgG theability of a scFv to interfere with interaction partners through sterichindrance is largely reduced. Last but not least it shall be noted thatdue to its bivalent mode of binding to the homo-trimeric TNFα, theaffinity of the parent IgG may have been reported too high (SPRartifact). Consequently, when comparing affinities between the parentalbivalent rabbit IgG and the humanized monovalent scFv, the reported“loss in affinity” may be overestimated.

1.2.3.1 Affinity

Affinity of humanized scFvs to human TNFα was determined by SPRmeasurements (see also 2.1.1). Affinity was determined using 2-foldserial dilutions of the respective scFvs. The scFvs were derived from arabbit monoclonal antibody. Two scFv variants were generated, named“CDR” (CDR) and “structural graft” (STR). To assess the relativecontribution of framework substitutions in the light and the heavy chainand to possibly reduce the number of rabbit amino acid residuesintroduced in the human framework, domain shuffling experiments wereperformed. Therefore, scFv constructs containing a CDR grafted lightchain and a structural grafted heavy chain (CDR/STR) were generated forclone 16-22-H05.

The top ranking scFvs 16-22-H05-sc02 (STR) and 16-22-H05-sc04 (CDR/STR)bound with affinities of 4.5×10⁻¹¹ and 1.1×10⁻¹⁰ M, respectively.16-22-H05-sc04 showed only a slight reduction of affinity when comparedto its “structural graft” variant (16-22-H04-sc02) (see Table 7). Theseresults suggest that affinity of the humanized scFvs mainly depends onthe few rabbit amino acids introduced into the human heavy chainframework.

1.2.3.2 Potency

The ability of the humanized scFvs to neutralize human TNFα was analyzedusing the L929 assay (see also 2.1.2). The potency (IC₅₀ and IC₉₀) toneutralize TNFα induced apoptosis was analyzed for 16-22-H5 derivedscFvs and compared to the potency of the reference antibody infliximabto allow for direct comparison of IC₅₀ and IC₉₀ values from differentassay plates. Relative IC₅₀ and IC₉₀ values were calculated in massunits (ng/ml) of infliximab and the scFvs. Potency analysis wasperformed several times on different days with different lots ofantibody fragments. FIG. 7 shows representative dose-response curvesfrom one experiment for each of the two scFvs. Mean values of replicatemeasurements are shown in Table 7 (standard deviations are summarized inthe legend of the table).

The humanized scFvs inhibited TNFα induced apoptosis with lower IC₅₀ andIC₉₀ values than infliximab (see Table 7). Consistent with SPR results,the domain shuffled variant 16-22-H05-sc04 (CDR/STR) exhibited equalpotency when compared to the structural graft 16-22-H05-sc02 (STR). ThescFvs 16-22-H05-sc04 and 16-22-H05-sc02 showed excellentTNFα-neutralizing activities, with IC₅₀ values of 14.6- and 13.1-foldbetter than infliximab, respectively. IC₉₀ values of 16-22-H05-sc04 and16-22-H05-sc02 were 13.1- and 12.6-fold better than for infliximab,respectively. As observed for the parental rabbit monoclonal antibodiesthere was no clear correlation between affinity and potency ofantibodies (correlation not shown). Nevertheless, scFv derived from16-22-H05 showing the highest affinities (16-22-H05-sc02 (STR) and16-22-H05-sc04 (CDR/STR)) also showed highest potency. Additionally,results from the neutralization assays suggest that a certain thresholdaffinity needs to be achieved for efficient inhibition of TNFαsignaling. For example, scFvs 16-14-D08-sc01 (CDR), 16-15-009-sc01(CDR), 16-24-H07-sc01 (CDR) and 17-20-G01-sc01 (CDR), all binding toTNFα with affinities above 1 nM, show poor potential to neutralize TNFα(not shown).

1.2.3.3 Species Cross-Reactivity (Cynomolgus and Rhesus Monkey TNFα)

Species cross-reactivity for the top ranking scFvs was determined by twomethods: 1) potency to neutralize Cynomolgus monkey and Rhesus monkeyTNFα in the L929 assay and 2) affinity to Cynomolgus monkey and Rhesusmonkey TNFα by SPR. The potency to neutralize TNFα from the differentspecies was determined by the L929 assay similarly as described abovefor human TNFα using Cynomolgus monkey and Rhesus monkey TNFα,respectively (see also 2.1.2). TNFα from both species showed verysimilar potency to induce L929 apoptosis (data not shown). Therefore,same concentrations of human and monkey TNFα were used for speciescross-reactivity testing. Additionally, binding kinetics (by SPR) toCynomolgus monkey and Rhesus monkey TNFα were determined using a similarassay as for human TNFα (see also 2.1.1).

All scFvs derived from the clone 16-22-H05 showed cross-reactivity toCynomolgus monkey and Rhesus monkey TNFα (see Table 7). The affinitieswere similar, namely 2.0×10⁻¹⁰ and 2.3×10⁻¹⁰ M for Cynomolgus monkey andRhesus monkey, respectively. The difference in affinity between humanand monkey TNFα was about 5-fold. Potencies to neutralize Cynomolgusmonkey, Rhesus monkey and human TNFα correlated well with the affinitiesto the respective TNFαs. Consequently the two clones derived from16-22-H05 showed between 5- to 7-fold lower potencies towards monkeyTNFα as compared to human TNFα (see Table 7 and FIG. 8). To summarize,the two scFvs showed species cross-reactivity to Cynomolgus and RhesusTNFα.

1.2.3.4 Blocking of the Human TNFα-TNFRI/II Interaction

In addition to the L929 assay, the potency of each humanized scFv toinhibit the interaction between human TNFα and TNFRI/II was assessed byELISA (see 2.1.3). Similarly to the L929 assay, individual IC₅₀ valueson each plate were calibrated against the IC₅₀ of the reference moleculeinfliximab that was taken along on each plate and relative IC₅₀ and IC₉₀values were calculated in mass units (ng/ml) of Infliximab and thescFvs.

Neutralization assays can distinguish potencies of target blockingantibodies only if they bind their target with an equilibrium bindingconstant (KD) that is higher than the target concentration used in thepotency assay (KD>target concentration). For the L929 assay a TNFαconcentration of 5 pM was used while in the TNFRI/II inhibition ELISAs aTNFα concentration of 960 pM was used. Therefore, theoretically, theL929 assay can differentiate potencies between scFvs with KD>5 pM, whilethe inhibition ELISA can only differentiate potencies between scFvs withKD>960 pM. Since all of the scFvs analyzed showed KDs below 960 pM,potencies between scFvs with different affinities (but similar mechanismof action) can be differentiated only in the L929 assay.

16-22-H5-sc02 and 16-22-05-sc04 showed potencies for blocking of theTNFα-TNFRI interaction between 2.8 and 3.5-fold higher compared toinfliximab while the potency compared to infliximab in the L929 assaywas significantly higher (13.1 and 14.6-fold). When comparing therelative IC₅₀ values for the parental rabbit IgG (see Table 2) with therelative IC₅₀ values for the humanized scFvs (Table 7) potencies of thescFvs are in general slightly higher compared to the parental IgGalthough affinities in general are in the same range for the parentalrabbit IgG. Since potencies of antibodies and scFvs were compared inmass units, the number of valencies (TNFα binding sites) at eachconcentration is about 2.9-fold higher for the monovalent scFvs comparedto the more than five-fold heavier but bivalent IgG. With veryhigh-affinity binding scFvs, this results in more potent blocking of theTNFα and TNFRI/II interaction because the lack of avidity is no longercritical for activity. In contrast, with low-affinity monovalent domainsthe opposite has been published (Coppieters et al. Arthritis &Rheumatism, 2006; 54:1856-1866). For the reasons mentioned above,results from the inhibition ELISA were not used for ranking of potenciesbetween the different antibodies but primarily for comparison of thepotential of antibodies to block the interaction with TNFRI versusTNFRII. The investigated scFvs blocked the interaction between both TNFαreceptors with comparable potencies (Table 9, FIG. 9 and FIG. 10).

1.2.3.5 Target Specificity (Selectivity for Binding to TNFα Versus TNFβ)

Specificity of the two scFvs (16-22-H05-sc02 and 16-22-H05-sc04) forTNFα over TNFβ was confirmed by assessment of the relative potential ofTNFβ as compared to TNFα to half-maximally inhibit TNFα binding to eachscFv and was measured in a competition ELISA (see also 2.1.4). Thequality of recombinant human TNFβ has been analyzed 1) for purity bySDS-page and HPLC analysis, and 2) for biological activity in the mouseL929 cytotoxicity assay, by the manufacturer of the protein. As shown inFIG. 11, the interaction between each of the scFvs with biotinylatedTNFα was blocked by unlabeled TNFα with IC₅₀ values ranging from 60 to260 ng/ml, while TNFβ did not show any significant effect even at thehighest concentration of TNFβ tested (1250 pg/ml). Hence, all of thescFvs analyzed bind specifically to TNFα but not to its closesthomologue, TNFβ. TNFβ did not show any significant inhibition of TNFαbinding to scFvs at the concentrations tested. Therefore, the TNFβconcentration required to half-maximally inhibit TNFα binding has to besignificantly higher than the highest concentration of TNFβ used in theassay (1250 pg/ml). When comparing concentrations of TNFα and TNFβrequired to half-maximally inhibit TNFα binding to the scFvs, theselectivity for binding to TNFα over TNFβ is significantly higher thanapproximately 5000 to 20,000 fold for all of the fragments tested (seealso Table 7). Therefore, off-target binding of any of the scFvs appearshighly unlikely.

The results of the experiments described above are summarized in tables7 to 9.

TABLE 7 In vitro binding and activity properties of humanized scFvs.CDR: “CDR graft”, STR: “structural graft”. Potency analysis wasperformed several times on different days with different lots ofantibody fragments with the following standard deviations:16-22-H05-sc02 (n = 3): rel. IC₅₀ = 13.1 ± 1.8 and rel. IC₉₀ = 12.6 ±3.5; 16-22-H05-sc04 (n = 2): rel. IC₅₀ = 14.6 ± 0.6 and rel. IC₉₀ = 13.1± 2.4. Affintiy to human TNFα Affintiy to cyno TNFα Affintiy to RhesusTNFα k_(a) k_(d) KD k_(a) k_(d) KD k_(a) k_(d) KD scFv Design (M⁻¹s⁻¹)(s⁻¹) (M) (M⁻¹s⁻¹) (s⁻¹) (M) (M⁻¹s⁻¹) (s⁻¹) (M) 16-22-H05-sc02 STR9.6E+05 4.3E−05 4.5E−11 4.9E+05 9.6E−05 2.0E−10 6.7E+05 1.5E−04 2.3E−1016-22-H05-sc04 CDR/STR 6.8E+05 7.1E−05 1.1E−10 nd nd Species-specificityTarget selectivity Potency IC₅₀ [ng/mL] rel. IC₅₀ of scFv rel. IC₅₀*rel. IC₉₀ ^(&) human cyno Rhesus TNFβ vs TNFα 16-22-H05-sc02 13.1 12.60.6 2.9 3.0 >>10′000 16-22-H05-sc04 14.6 13.1 0.5 3.5 2.9 >>10′000*IC_(50, Infliximab) (ng/mL)/IC_(50, scFv) (ng/mL)^(&)IC_(90, Infliximab) (ng/mL)/IC_(90, scFv) (ng/mL)

TABLE 8 Specifications of humanized scFvs of the present invention.Stability Expression Producibility Monomer loss after Monomer loss afterExpression Refolding Thermal 4 w at 10 g/L at 5 freeze/thaw yield yieldunfolding 4° C. (−65, −20) cycles [g/L] [mg/L] [° C.] [Δ %] [Δ %]16-22-H05-sc02 0.26 29.8 71.3 0.2 (0.0, 0.0) 0.0 16-22-H05-sc04 0.2624.1 72.6 0.4 (0.2, 0.1) 0

TABLE 9 Potency of scFvs to block the TNFα-TNFR1 and TNFα-TNFR2interaction. Blocking of TNFα-TNFRI interaction Blocking of TNFα-TNFRIIinteraction rel. rel. IC₅₀ IC₉₀ rel. rel. IC₅₀ IC₉₀ scFv IC₅₀* IC₉₀ ^(&)[ng/mL] [ng/mL] IC₅₀* IC₉₀ ^(&) [ng/mL] [ng/mL] 16-22-H05-sc02 3.5 2.413.9 39.8 5.7 3.3 20.0 56.9 16-22-H05-sc04 2.8 2.1 17.7 45.3 3.5 1.532.6 127 *IC_(50, Infliximab) (ng/mL)/IC_(50, scFv) (ng/mL)^(&)IC_(90, Infliximab) (ng/mL)/IC_(90, scFv) (ng/mL)

2. Methods

2.1 Lead Characterization Assays

2.1.1 Binding Kinetics and Species Cross-Reactivity by SPR

Binding affinities of scFvs towards human TNFα were measured by surfaceplasmon resonance (SPR) using a MASS-1 SPR instrument (Sierra Sensors).Performance of the SPR assay was qualified by analysis of a referenceantibody antigen interaction such as certolizumab-TNFα interaction. Thepegylated Fab-fragment certolizumab was selected as reference due to itsmonovalent binding mode similar to that of scFvs. Using the same assaysetup as for affinity measurements of the scFvs, a value of 9.94×10⁻¹¹ Mwas determined for the affinity of certolizumab to TNFα. This value isin good agreement with published K_(D) values of 9.02±1.43×10⁻¹¹ M (BLAcertolizumab; BLA number: 125160; submission date: Apr. 30, 2007).

For affinity measurements of scFvs human TNFα (Peprotech, Cat. No.300-01) was immobilized on a sensor chip (SPR-2 Affinity Sensor, Amine,Sierra Sensors) by amine-coupling to reach an immobilization level of 50to 100 RUs (immobilization levels achieved during SPR analysis werebetween 40 to 120 RUs). In a first step, affinity screening of scFvs wasperformed using only one scFv concentration (90 nM). In a second step,for the best performing scFvs, Single Injection Cycle Kinetics (SiCK)were measured from a single injection cycle by simultaneously injectingsix analyte samples at different concentrations into each of the eightparallel channels in the MASS-1 system. For affinity screenings,humanized scFvs were injected into the flow cells at a concentration of90 nM for three minutes and dissociation was monitored for 12 minutes.For the subsequent more precise affinity determinations, two-fold serialdilutions of scFv ranging from 45 to 1.4 nM were injected into the flowcells for three minutes and dissociation of the protein from TNFαimmobilized on the sensor chip was allowed to proceed for 12 minutes.The apparent dissociation (k_(d)) and association (k_(a)) rate constantsand the apparent dissociation equilibrium constant (K_(D)) werecalculated with the MASS-1 analysis software (Analyzer, Sierra Sensors)using one-to-one Langmuir binding model and quality of the fits wasmonitored based on Chi², which is a measure for the quality of the curvefitting. The smaller the value for the Chi² the more accurate is thefitting to the one-to-one Langmuir binding model. For affinityscreenings, results were deemed valid if the Chi² was below 10 for theconcentration analyzed. In cases where several scFv concentrations wereanalyzed, results were deemed valid if the average Chi² over all theconcentrations tested was below 10. Acceptance criteria were met for allscFvs tested.

Species cross-reactivity to Cynomolgus monkey (Sino Biological, Cat. No.90018-CNAE) and Rhesus monkey (R&D Systems, Cat. No. 1070-RM-025/CF)TNFα (Peprotech, Cat. No. 315-01A) was measured using the same assaysetup and applying the same quality measures as described above forhuman TNFα. For Cynomolgus and Rhesus monkey TNFα immobilization levelsranging from 50 to 180 RUs and from 90 to 250 RUs, respectively, wereachieved. The scFvs were analyzed using two-fold serial dilutions withconcentrations ranging from 45 to 1.4 nM. The average Chi² values werebelow 10 for all of the scFvs tested.

2.1.2 TNFα-Induced Apoptosis in L929 Fibroblasts (Neutralization ofHuman, Non-Human Primate and TNFα by scFvs)

The ability of scFvs to neutralize the biological activity ofrecombinant human TNFα was assessed using mouse L929 fibroblasts(ATCC/LGC Standards, Cat. No. CCL-1). L929 cells were sensitized toTNFα-induced apoptosis by addition of 1 pg/ml actinomycin D. Three-foldserial dilutions of anti-TNFα reference antibody or scFvs (3000-0.05ng/ml) and 5 pM recombinant human TNFα (Peprotech, Cat. No. 300-01) werepre-incubated at room temperature for 1 hour. The used TNFαconcentration (5 pM) induces submaximal L929 apoptosis (EC₉₀). Afteraddition of the agonist/inhibitor mixtures the cells were incubated for24 hours. Survival of the cells was determined by a colorimetric assayusing the WST-8(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt) cell proliferation reagent (Sigma Aldrich, Cat. No.96992). WST-8 is reduced by cellular dehydrogenases to an orangeformazan product. The amount of formazan produced is directlyproportional to the number of living cells. Data were analyzed using afour-parameter logistic curve fit using the Softmax Data AnalysisSoftware (Molecular Devices), and the concentration of referenceantibody or scFvs required to neutralize TNFα-induced apoptosis by 50%and 90% (IC₅₀ and IC₉₀) was calculated (see also FIG. 7). In order torender IC₅₀ and IC₉₀ values directly comparable between experiments thatwere performed on different days or on different assay plates, IC₅₀ andIC₉₀ values were calibrated against the reference antibody infliximab.To control precision of the response, the dose-response curves wereanalyzed in duplicate. Standard deviations and CVs were calculated foreach measurement point (CV<20%).

Species cross-reactivity to Cynomolgus monkey (Sino Biological, Cat. No.90018-CNAE) and Rhesus monkey (R&D Systems, Cat. No. 1070-RM-025/CF)TNFα was measured using the same assay setup and applying the samequality measures as described above for human TNFα. Similarly to thehuman counterpart, TNFα concentrations that induce submaximal L929apoptosis (EC₉₀) were used for species cross-reactivity testing. TNFαfrom both species showed very similar potency to human TNFα to induceL929 mouse fibroblast apoptosis. Consequently the same concentration ofTNFα (5 pM) was used for oth species tested. During speciescross-reactivity testing CVs of most of the duplicate measurement pointswere below 10%.

2.1.3 TNFα Inhibition ELISA

The inhibitory effect of scFvs on ligand binding was assessed using anELISA, a biochemical method solely reproducing the interaction betweenTNFα and TNFRI and TNFRII.

For the first inhibition ELISA, the extracellular domain of TNFRI fusedto the Fc region of human IgG (R&D Systems, Cat. No. 372-RI) was coatedon a 96-well Maxisorp ELISA at a concentration of 0.5 pg/ml. For thesecond inhibition ELISA, the extracellular domain of TNFRII fused to theFc region of human IgG (R&D Systems, Cat. No. 726-R2) was coated at aconcentration of 2 pg/ml. All subsequent steps were identical for bothassays. In order to detect binding of TNFα to TNFRI and TNFRII, TNFα wasbiotinylated prior to its use. Biotinylated human TNFα (960 pM, 50ng/ml) was first incubated with 3-fold serially diluted humanizedanti-TNFα scFvs and infliximab (10′000 ng/ml-0.2 ng/ml) for 1 hour atroom temperature. The TNFα/antibody fragment mixtures were transferredto the TNF receptor immobilized plates and binding of unblocked TNFα tothe immobilized TNFα receptor was detected after incubation at roomtemperature for 20 minutes with the biotin-binding streptavidin-HRP (SDTReagents, Cat. No. SP40C). Addition of 3′,5,5′-tetramethylbenzidine(TMB) substrate resulted in a colorimetric read-out that wasproportional to the binding of TNFα to TNFRI and TNFRII. Before use inthe competition ELISA, the biological activity of the biotinylated TNFαwas confirmed in the L929 assay. The EC₅₀ of biotinylated TNFα wassimilar to the EC₅₀ of unlabeled TNFα (data not shown). Similar to theL929 assay described above, data were analyzed using a four-parameterlogistic curve fit using the Softmax Data Analysis Software (MolecularDevices), and the concentration of scFvs required to inhibit interactionof TNFα and TNFR by 50% and 90% (IC₅₀ and IC₀₀) was calculated. In orderto render IC₅₀ and IC₉₀ values directly comparable between experimentsthat were performed on different days or on different assay plates, IC₅₀and IC₉₀ values were calibrated against the reference antibodyinfliximab.

To control precision of the response, the dose-response curves wereanalyzed in duplicate. Standard deviations and CVs were calculated foreach measurement point (CV<25%).

2.1.4 Target Specificity

To confirm specificity of the anti-TNFα scFvs, binding to the mosthomologous family member TNFβ was assessed. The potential to inhibit theinteraction of biotinylated TNFα with scFvs by unlabeled TNFβ(Peprotech, Cat. No. 300-01B) and TNFα (Peprotech, Cat. No. 300-01) wasanalyzed by competition ELISA. For this purpose, the scFvs were coatedon a 96-well Maxisorp ELISA plate at a concentration of 1 pg/ml. Bindingof biotinylated TNFα (75 ng/ml) to the coated scFvs in presence of5-fold serially diluted unlabeled TNFα (50 pg/ml-0.00013 pg/ml) or TNFβ(1250 pg/ml-0.00013 pg/ml) was detected using the biotin-bindingstreptavidin-HRP (SDT Reagents, Cat. No. SP40C) as described above. Forthe dose-response curve with TNFα data were analyzed using afour-parameter logistic curve fit using the Softmax Data AnalysisSoftware (Molecular Devices), and the concentration of unlabeled TNFαrequired to block the interaction of biotinylated TNFα with the coatedscFv by 50% (IC₅₀) was calculated. TNFβ did not show any significantinhibition of the interaction between biotinylated TNFα and scFvs (seealso FIG. 11). To quantify the relative potential of TNFβ as compared toTNFα to inhibit TNFα binding to each scFv the IC₅₀ to inhibit theinteraction by TNFβ relative to TNFα was calculated. Since nosignificant inhibition was observed when using TNFβ at an approximately5′000 to 20′000-fold higher concentration than the IC₅₀ of TNFα, theselectivity for binding to TNFα over TNFβ was determined to besignificantly higher than 5′000 to 20′000-fold. To control precision ofthe response, the dose-response curves were analyzed in duplicate.Standard deviations and CVs were calculated for each measurement point(CV<25% for all but one of the TNFα/β concentrations tested). All scFvfulfilled this criterion.

2.1 CMC Analytics

2.2.1 Reducing SDS-PAGE

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) isan analysis technique used for qualitative characterization and tocontrol purity of proteins. According to the United States Pharmacopeia(USP) (USP Chapter 1056) analytical gel electrophoresis is anappropriate and routine method to identify and to assess the homogeneityof proteins in drug substances.

The method is used to quantify the amount of scFv product from E. colilysates to derive the expression yield after fermentation. Anotherapplication of the method is to verify the identity of test substancesbased on their molecular weight with respect to the theoretical values.For supportive purposes this method is used to quantify the purity oftest samples with respect to process-related impurities (host cellproteins) and product related impurities (degradation products oradducts).

The SDS-PAGE analyses were performed with commercially available precastgel system “Mini Protean” obtained from Bio-Rad Laboratories Inc.Humanized scFvs were analyzed on “Any kD” resolving gels (#456-9036). Inboth cases the Tris/Glycine buffer system recommended by themanufacturer was used. For the detection of protein bands eithercoomassie staining with SimplyBlue™ staining solution (Life TechnologiesCorp., #LC6060) or silver staining with the Pierce Silver Stain Kit(Thermo Fisher Scientific Inc., #24612) was employed. For the stainingprocedures the protocols of the respective supplier were followed.

The documentation and analysis of the stained protein gels was performedwith the documentation system ChemiDoc XRS System (Bio-Rad LaboratoriesInc., #170-8265) and software Image Lab, Version 4.0.1 (Bio-RadLaboratories Inc., #170-9690).

Titer Determination of Lysate Samples

SDS-PAGE allows for specific detection of the protein of interest in themixture of host cell proteins. A reference standard dilution series inthe linear range of the method (which was determined in advance) wasincluded on every gel. A linear regression of band intensities (measuredby densitometry) versus nominal concentrations of the reference standardwere used to calculate a standard curve, which in turn, was used toextrapolate scFv content in the sample.

The lysate samples of unknown product concentrations were loaded indifferent dilutions (at least 1:10 in dilution buffer) to have at leastone scFv concentration in the linear range of the method. The productamount was calculated based on the measured band intensities of the scFvand the concentration was determined using the dilution factors of thesample preparation. The values were averaged for all samples that werewithin the linear range of the standard curve.

As an additional test of the suitability of the method for thequantification of lysate sample an inhibition/enhancement test wasperformed by spiking a lysate sample with a known amount of referencestandard. Calculation of the spike recovery at a sample dilution of 1:10in dilution buffer resulted in a value of 95.4% which is at the samelevel of precision as observed with the reference standard in dilutionbuffer. Thus, no significant matrix interference in cell lysates wasobserved and the method was deemed suitable for the quantification ofscFv content in cell lysates.

Protein Purity and Content

To show the suitability of the method to determine the content andthereby also the purity of test samples, the lower limit of detection(LOD) for a reference scFv was determined visually (by identifying theprotein band) at a nominal load of 0.02 pg, the evaluation of theintensity histogram of the respective lane shows a signal-to-noise ratioat this load of approximately 2. In addition, the linear range for thequantification was determined by analyzing the main bandsdensitometrically.

The fit of the data with a linear regression, results in a coefficientof determination (R²) of 0.9998, thus indicating a good quality of thefit. In addition to the overall quality of the fit the relative error ofeach individual data point was determined to document the suitability ofthe method in the chosen range. The relative errors are below 10% forall data points indicating good accuracy of this method.

2.2.2 UV Absorbance at 280 nm

The method UV absorbance at 280 nm is a total protein assay as outlinedin USP Chapter 1057. Protein solutions absorb UV light at a wavelengthof 280 nm due to the presence of aromatic amino acids. The UV absorbanceis a function of the content of tyrosine and tryptophan residues in theprotein and is proportional to the protein concentration. The absorbanceof an unknown protein solution can be determined according to USPChapter 851 on spectroscopy by applying Beer's law: A=erc, where theabsorbance (A) is equal to the product of the molar absorptivity (ε),the absorption path length and the concentration of the substance. Themolar absorptivity for the scFv was calculated with the software VectorNTI® (Life Technologies Corporation).

The measurement of the UV absorbance was performed with the Infinityreader M200 Pro equipped with Nanoquant plate (Tecan Group Ltd.). Theabsorbance of the protein samples were measured at 280 nm and 310 nm,where the latter wavelength was serving as a reference signal that wassubtracted from the 280 nm signal. To account for potential interferenceof the sample matrix a blank subtraction was performed for eachmeasurement.

The final absorbance signal of a protein sample obtained was used tocalculate the protein concentration using Lambert-Beer's law.

All measurements were performed within the range given by theinstruments specifications in the measurement range of 0-4 OD, where areproducibility of <1% and a uniformity of <3% is specified by themanufacturer.

2.2.3 SE-HPLC (Size Exclusion High-Pressure Liquid Chromatography)

SE-HPLC is a separation technique based on a solid stationary phase anda liquid mobile phase as outlined by the USP chapter 621. This methodseparates molecules based on their size and shape utilizing ahydrophobic stationary phase and aqueous mobile phase. The separation ofmolecules is occurring between the void volume (V₀) and the totalpermeation volume (V_(T)) of a specific column. Measurements by SE-HPLCwere performed on a Chromaster HPLC system (Hitachi High-TechnologiesCorporation) equipped with automated sample injection and a UV detectorset to the detection wavelength of 280 nm. The equipment is controlledby the software EZChrom Elite (Agilent Technologies, Version 3.3.2 SP2)which also supports analysis of resulting chromatograms. Protein sampleswere cleared by centrifugation and kept at a temperature of 6° C. in theautosampler prior to injection. For the analysis of scFv samples thecolumn Shodex KW402.5-4F (Showa Denko Inc., #F6989201) was employed witha standardized buffered saline mobile phase (50 mM Sodium acetate pH6.0, 250 mM sodium chloride) at the recommended flow rate of 0.35mL/min. The target sample load per injection was 5 pg. Samples weredetected by an UV detector at a wavelength of 280 nm and the datarecorded by a suitable software suite. The resulting chromatograms wereanalyzed in the range of V₀ to V_(T) thereby excluding matrix associatedpeaks with >10 min elution time.

To ensure intermediate precision of the method, a reference standard wasroutinely measured at the beginning and end of each HPLC sequence. Thereference standard used for this system suitability test was a scFv thathad been produced as a batch and was aliquoted to be used for eachmeasurement timepoint.

2.2.4 DSF (Differential Scanning Fluorimetry)

The method DSF is a non-compendial method to measuretemperature-dependent protein unfolding. The measurement of the thermalunfolding temperature by DSF were performed with a MX3005P qPCR machine(Agilent Technologies) controlled with the MX Pro software package(Agilent Technologies) and equipped with an excitation/emission filterset at 492/610 nm. The reactions were set-up in Thermo fast 96 white PCRplates (Abgene; #AB-06001W). For the detection of protein unfolding acommercially available stock solution of the dye SYPRO orange (MolecularProbes; #S6650) was used at a final dilution of 1:1′000. The proteinsamples were diluted for the unfolding measurements to a finalconcentration of 50 pg/mL in a standardized buffered saline solution.The thermal unfolding was performed by a temperature program starting at25° C. ramping up to 96° C. in 1° C. steps with a duration of 30seconds. During the temperature program the fluorescence emission ofeach sample was recorded. The recorded raw data was processed andevaluated with a package of Microsoft Excel templates (Niesen, NatureProtocols 2007, Vol. 2 No. 9) and the fluorescence data was fitted witha Boltzmann equation using the program GraphPad Prism (GraphPadSoftware, Inc.) to obtain the midpoint of transition (T_(m)).

In order to produce reliable and robust measurements of the midpoint ofunfolding at least duplicate measurements were performed. With respectto the data quality only measurements with a goodness of fit (R²)>0.9900and a 95% confidence interval of the T_(m) of smaller than 0.5% wereconsidered.

For an assessment of the intermediate precision a reference standard(known characterized scFv) was included with every measurement to allowfor comparison of assay performance on different days.

2.2.5 Stability Study

In order to assess the stability of different scFv constructs as aread-out for the developability of these molecules a short-termstability study protocol was designed. The protein constructs wereconcentrated in a simple buffered saline formulation (see above) to thetarget concentrations of 1 and 10 mg/mL. The monomer content wasdetermined by SE-HPLC to confirm that the purity is exceeding thesuccess criteria of >95%. Subsequently the protein samples were storedat <−65, −20, 4 and 37° C. for the duration of 4 weeks and aliquots wereanalyzed at various time points. The primary read-out is the analysis bySE-HPLC, which allows the quantification of soluble higher molecularweight oligomers and aggregates. As supportive measurements the proteincontent is determined by UV absorbance at 280 nm, which gives anindication whether during the storage period substantial amounts ofprotein were lost by precipitation. For the storage screw cap tubes wereused (Sarstedt, Cat. No. 72.692.005) with filling amounts of 30-1500 pgper aliquot. Additionally purity is determined by SDS-PAGE thatindicates the stability of the construct with respect to degradation orcovalent multimerization.

Example 3: Generation of Humanized Diabody and IgG

The single-chain diabody construct was designed by arranging thevariable domains in a VLA-L1-VHB-L2-VLB-L3-VHA configuration. In theseconstructs the VLA and VHA and VLB and VHB domains jointly form thebinding site for TNFα. The peptide linkers L1-L3 connecting the variabledomains were constructed of glycine/serine repeats. The two shortlinkers L1 and L3 are composed of a single G₄S repeat, whereas the longlinker L2 is composed of the sequence (G₄S)₄. The nucleotide sequencesencoding the humanized variable domains (Example 2; 1.2.1.) were de novosynthesized and cloned into an adapted vector for E. coli expressionthat is based on a pET26b(+) backbone (Novagen). The expression andpurification was performed as described for the scFvs in Example 2;1.2.1.

The humanized IgG was constructed by cloning the variable domains asuitable mammalian expression vector for transient heterologousexpression containing a leader sequence and the respective constantdomains e.g. the pFUSE-rIgG vectors (Invivogen). The transientexpression of the functional IgG was performed by co-transfection ofvectors encoding the heavy and light chains with the FreeStyle™ MAXsystem in CHO S cells. After cultivation for several days thesupernatant of the antibody secreting cells was recovered forpurification. Subsequently the secreted IgGs were affinity purified byProtein A sepharose (GE Healthcare). The elution fractions were analyzedby SDS-PAGE, UV absorbance at 280 nm and SE-HPLC.

The affinities of the antibody molecules were determined using a Biacoreinstrument as described in Example 2 under 2.1.1).

TABLE 10 k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) IgG 1.90 × 10⁶ 7.92 × 10⁻⁵4.17 × 10⁻¹¹ scDb 9.40 × 10⁵ 2.02 × 10⁻⁵ 2.15 × 10⁻¹¹ scFv 8.93 × 10⁵3.38 × 10⁻⁵ 3.79 × 10⁻¹¹

The potencies of the antibody molecules were determined in an L929 assay(the method is described in Example 2 under 2.1.2).

TABLE 11 Potency IC₅₀ (nM) IgG 0.02 scDb 0.01 scFv 0.03

Example 4: Determination of Stoichiometry of TNFα Binding

The binding stoichiometry of 16-22-H5 to TNFα was determined usingSE-HPLC. 16-22-H5-scFv and TNFα were incubated at two different molarratios, namely at a 1:1 and 4.5:1 molar ratio. Since TNFα exists as atrimer in solution the indicated molar ratios refer to theTNFα_(trimer). Thus, in the 4.5:1 ratio the 16-22-H5-scFv is in excessand should occupy all TNFα_(trimer) binding positions resulting incomplexes of 1 TNFα_(trimer) with 3 scFv. However, under equimolarconditions there is not enough scFv present to saturate all 3theoretical TNFα binding sites. Therefore, also complex variants withless than 3 scFv bound are expected. TNFα and 16-22-H5-scFv wereincubated for 2 hours at RT to allow for complex formation. Samples werethen centrifuged at 4° C. for 10 min. 10 μL of each sample were analysedon SE-HPLC. The SE-HPLC analysis was performed with 50 mM phosphatebuffer pH 6.5, 300 mM NaCl as eluent at a flow rate of 0.35 mL/min.Eluted protein peaks were detected at a wavelength of 280 nm. The columnwas calibrated using the Gel filtration Calibration Kit from GEHealthcare (LMW, HMW) in advance for the determination of apparentmolecular weights.

The bottom panel of FIG. 12 shows the elution profile with equimolaramounts of scFv and TNFα which is overlayed with the profiles ofTNFα_(trimer) alone and scFv alone. Due to the trimerization of the TNFαin solution there are theoretically up to three equivalent binding sitesfor the scFv present on each trimer and hence the scFv molecules arelimiting. Under these conditions all three complex species (3:1, 2:1,1:1) were identified. The top panel of FIG. 12 shows the elution profileof the complex with excess amounts of scFv. The surplus of unbound scFveluted at the expected retention time. The TNFα peak was quantitativelyconsumed for complex formation and disappeared completely. The peak ofthis complex shifted towards lower retention times, and correlated wellwith the retention time of the peak with the largest molecular weight ofthe equimolar setup. For this reason it was concluded that all availablebinding sites on the TNFα were occupied by scFv and thus, the bindingstoichiometry is 3:1 (scFv:TNFα) if the scFv is available in excess.

Further to these qualitative observations, the apparent bindingstoichiometry was also calculated based on the apparent MW of the16-22-H5-scFv:TNFα complex as determined by SE-HPLC. Based on retentiontime, the apparent MW was calculated to be 139.7 kDa. According toequation (1) below the apparent binding stoichiometry was calculated tobe 3.3. This correlates well with the theoretical number of threeequivalent binding sites available for scFv on the TNFα_(trimer) and theobservations above where a 3:1 binding stoichiometry was determined.

$\begin{matrix}{{{{binding}\mspace{14mu} {stochimetry}\mspace{14mu} \left( {{scF}{v:{TNF\alpha}}} \right)} = \frac{{{MW}\left( {{complex}\mspace{14mu} {app}} \right)} - {M{W\left( {{TNF}\; \alpha \mspace{14mu} {theo}} \right)}}}{M{W\left( {{scFv}\mspace{14mu} {theo}} \right)}}}\begin{matrix}{{{MW}\left( {{complex}\mspace{14mu} {app}} \right)}:} & {139.7{kDa}} \\{{{MW}\left( {{TNF}\; \alpha \mspace{14mu} {theo}} \right)}:} & {52.2{kDa}} \\{{{MW}\left( {{scFv}\mspace{14mu} {theo}} \right)}:} & {26.5{kDa}}\end{matrix}} & {{Equation}\mspace{14mu} (1)}\end{matrix}$

Example 5: Formation of TNFα:Antibody Complexes (Cross-Linking of TNFα)

The ability of the 16-22-H5-scDb to bind simultaneously to two TNFαmolecules was tested on a Biacore T200 instrument in HEPES buffercontaining 10 mM HEPES, 150 mM NaCl and 0.05% Tween. Biotinylated TNFα(Acro Biosystems) was captured via a biotinylated ssDNA oligo using theBiotin CAPture kit (GE Healthcare) according to the manufacturer'sinstructions. 0.25 pg/mL of biotinylated TNFα were injected with a flowrate of 10 μL/min for 3 min to reach a capture level of approximately200 to 300 RUs (resonance units). The antibodies 16-22-H5-scDb and16-22-H5-scFv, as a control, were injected over the TNFα immobilizedsurface for 2 min at a flow rate of 30 μL/min at a concentration of 90nM. Following association of the antibody fragments, TNFα (Peprotech)was injected for 5 min with a flow rate of 30 μL/min at 90 nM. Theantibody and TNFα concentrations were selected close to saturation ofthe binding. The measurement was performed at 25° C. FIG. 13 illustratesthat the bivalent 16-22-H5-scDb is able to bind two TNFα moleculessimultaneously while, as expected, the monovalent 16-22-H5-scFv bindsonly to one TNFα molecule.

Further, the formation of TNFα-antibody complexes was assessed atdifferent ratios of TNFα and 16-22-H5 antibody formats using SE-HPLC.16-22-H5-IgG (150 kDa) and 16-22-H5-scDb (52 kDa) were incubated withTNFα (52 kDa) at different molar ratios (1:3, 1:1, 3:1) in respect tobinding sites. Thus, IgG and scDb have 2 and TNFα has 3 binding sites.The antibody-TNFα mixtures were incubated for at least 30 min at 37° C.,cooled down for 10 min at RT and stored overnight at 2−8° C. Five to 10uL of protein mixture at a concentration of approx. 1 mg/mL wereinjected onto a TOSHO TSKgel UP-SW3000 column. The analysis wasperformed with 150 mM phosphate buffer pH 6.8, 100 mM NaCl as eluent ata flow rate of 0.3 mL/min. Eluted protein peaks were detected at awavelength of 214 nm. The column was calibrated using the BEH450 SECprotein standard mix (Waters) in advance for the determination of theapproximate molecular weights of the complexes. FIG. 14A shows theformation of 16-22-H5-IgG:TNFα complexes. Complexes that are ≥600 kDaindicate the formation of complexes consisting of ≥2 TNFα and ≥3 IgGmolecules. FIG. 14B shows the formation of 16-22-H5-scDb:TNFα complexes.Complexes that are ≥300 kDa indicate the formation of complexesconsisting of ≥2 TNFα and ≥3 scDb molecules.

Example 6: Inhibition of Cell Proliferation

The capacity of different antibody formats of 16-22-H5 and adalimumab toinhibit the proliferation of peripheral blood mononuclear cells (PBMC)was tested in a mixed lymphocyte reaction (MLR). PBMC from 2 healthydonors were cultured (RPM11640) in a 1:1 ratio in 96-well plates for 48h at 37° C./5% CO₂. After activation, cells were treated with anti-TNFαantibodies or IgG control antibody (all at a final concentration of 10pg/mL) in sextuplicates for another 5 d at 37° C./5% CO₂. 24 h beforethe end of incubation BrdU (20 uL/well) was added to each well andproliferation was determined by measuring BrdU uptake using acommercially available cell proliferation ELISA (Roche Diagnostics). Thestimulation index was determined by calculating the ratio of BrdU uptakebetween the antibody treated cells and mitomycin C (25 ng/mL) treatedcells. Table 12 and FIG. 15 illustrate that all tested antibody formatsof 16-22-H5 significantly inhibited T-cell proliferation comparable toadalimumab.

TABLE 12 concentration Stimulation Index (μg/ml) mean SD IgG control 105.2 0.5 Adalimumab 10 2.6** 0.6 16-22-H5-IgG 10 2.5** 0.6 16-22-H5-scDb10 4.1* 0.7 16-22-H5-scFv 10 4.0** 0.6 *p < 0.05; **p < 0.01

Example 7: Inhibition of LPS-Induced Cytokine Secretion

CD14⁺ monocytes in RPM11640 were seeded in 96-well plates and incubatedfor 16 h at 37° C./5% CO₂ in a humidified incubator. Then cells weretreated with anti-TNFα antibodies or IgG control antibody in duplicatesfor 1 h using final antibody concentrations ranging from 2 to 2000ng/mL. The monocytes were washed 3 times with cell culture medium andsubsequently incubated with LPS (100 ng/mL) for 4 h at 37° C./5% CO₂.IL-1β and TNFα concentrations in the cell culture supernatants weredetermined using commercially available ELISA kits (R&D Systems). TheResults are shown in Tables 13 and 14 and FIGS. 16A and B. IC₅₀ wasdetermined using a four-parameter logistic curve fit. Regardingsecretion of IL-1β the IC₅₀ values for 16-22-H5-IgG, 16-22-H5-scDb,16-22-H5-scFv and adalimumab is summarized in Table 13 below.

TABLE 13 Secretion of IL-1β IC₅₀ (pg/mL) IC₅₀ (nM) Adalimumab 121.3 0.8116-22-H5-IgG 81.55 0.54 16-22-H5-scDb 30.51 0.59 16-22-H5-scFv 16.980.65

As regards TNFα secretion, the determined IC₅₀ values for 16-22-H5-IgG,16-22-H5-scDb, 16-22-H5-scFv are summarized in Table 14.

TABLE 14 Secretion of TNFα IC₅₀ (pg/mL) IC₅₀ (nM) Adalimumab 174.0 1.1616-22-H5-IgG 120.5 0.80 16-22-H5-scDb 17.18 0.33 16-22-H5-scFv 13.480.52

TABLE 15 Vκ1 consensus sequences (rearranged) Positions SEQ according IDto Kabat: NO: Sequence Framework  1 to 23 56 DIQMTQSPSSLSASV I GDRVTITCFramework 35 to 49 57 WYQQKPGKAPKLLIY II Framework 57 to 88 58GVPSRFSGSGSGTDF III TLTISSLQPEDFATY YC

TABLE 16 Vλ germline-based framework IV sequences SEQ ID NO: Sequence 59FGTGTKVTVL 60 FGGGTKLTVL 61 FGGGTQLIIL 62 FGSGTKVTVL

1. An antibody or a functional fragment thereof capable of binding tohuman tumor necrosis factor alpha (TNFα), wherein said antibody orfunctional fragment comprises: i. a V_(L) domain comprising a CDR1region having an amino acid sequence in accordance with the amino acidsequence as shown in SEQ ID NO:1, a CDR2 region having an amino acidsequence in accordance with the amino acid sequence as shown in SEQ IDNO:2, and a CDR3 region having an amino acid sequence in accordance withthe amino acid sequence as shown in SEQ ID NO:3, and ii. a V_(H) domaincomprising a CDR1 region having an amino acid sequence in accordancewith the amino acid sequence as shown in SEQ ID NO:4, a CDR2 regionhaving an amino acid sequence in accordance with the amino acid sequenceas shown in SEQ ID NO:5, and a CDR3 region having an amino acid sequencein accordance with the amino acid sequence as shown in SEQ ID NO:6. 2.The antibody or functional fragment of claim 1, wherein said antibody orfunctional fragment comprises: i. a V_(L) domain comprising a CDR1region having the amino acid sequence as shown in SEQ ID NO:7, a CDR2region having the amino acid sequence as shown in SEQ ID NO:8, and aCDR3 region having the amino acid sequence as shown in SEQ ID NO:9, andii. a V_(H) domain comprising a CDR1 region having the amino acidsequence as shown in SEQ ID NO:10, a CDR2 region having the amino acidsequence as shown in SEQ ID NO:11, and a CDR3 region having the aminoacid sequence as shown in SEQ ID NO:12.
 3. The antibody or functionalfragment of claim 1 or 2, wherein said antibody or functional fragment:(i) binds to human TNFα with a dissociation constant (K_(D)) of lessthan 125 pM; (ii) is cross-reactive with Macaca mulatta (Rhesus) TNFαand with Macaca fascicularis (Cynomolgus) TNFα; (iii) has a greaterpotency to inhibit TNFα-induced apoptosis than infliximab, as determinedby an L929 assay; (iv) comprises a variable domain having a meltingtemperature, determined by differential scanning fluorimetry, of atleast 70° C. and/or (v) is capable of binding to human TNFα_(Trimer) ina stoichiometry (antibody: TNFα_(Trimer)) of at least
 2. 4. The antibodyor functional fragment of claim 1, wherein said antibody binds to humanTNFα with a K_(D) of less than 50 pM.
 5. The antibody or functionalfragment of claim 1, wherein said antibody or functional fragmentcomprises a V_(H) domain having the amino acid sequence as shown in SEQID NO:13.
 6. The antibody or functional fragment of claim 1, whereinsaid antibody or functional fragment comprises a V_(L) domain having anamino acid sequence selected from SEQ ID NO:14 and SEQ ID NO:54.
 7. Theantibody or functional fragment of claim 1, wherein said antibody orfunctional fragment is a single-chain variable fragment (scFv).
 8. Theantibody or functional fragment of claim 7, wherein said scFv has theamino acid sequence as shown in SEQ ID NO:15 or SEQ ID NO:55.
 9. Theantibody of claim 1, wherein said antibody is an immunoglobulin G (IgG).10. An antibody or functional fragment thereof, wherein said antibody orfunctional fragment binds essentially the same epitope as the antibodyor functional fragment of claim
 8. 11. The antibody or functionalfragment of claim 1, wherein the sum of (i) the number of amino acids inframework regions I, II, and III of the variable light domain of saidantibody or functional fragment that are different from the respectivehuman Vκ1 consensus sequences SEQ ID NOs: 56, 57, and 58, and (ii) thenumber of amino acids in framework region IV of the variable lightdomain of said antibody or functional fragment that are different fromthe most similar human λ germline-based sequence selected from SEQ IDNOs: 59, 60, 61, and 62, is less than
 7. 12. The antibody or functionalfragment of claim 1, wherein the framework regions I, II, and to III ofthe variable light domain of said antibody or functional fragmentconsist of human Vκ1 consensus sequences with SEQ ID NOs:56, 57, and 58,respectively, and framework region IV consists of a λ germline-basedsequence selected from among SEQ ID NOs:59, 60, 61, and
 62. 13. Anucleic acid encoding the antibody or functional fragment of claim 1.14. A vector or plasmid comprising the nucleic acid of claim
 13. 15. Acell comprising the nucleic acid of claim 13 or a vector or plasmidcomprising the nucleic acid of claim
 13. 16. A method of preparing theantibody or functional fragment of claim 1, comprising the steps of:culturing the as cell comprising a plasmid comprising said nucleic acida medium under conditions that allow expression of the nucleic acidencoding the antibody or functional fragment, and recovering theantibody or functional fragment from the cells or from the medium.
 17. Apharmaceutical composition comprising the antibody or functionalfragment of claim 1 in combination with an optional and optionally apharmaceutically acceptable carrier and/or excipient.
 18. A method oftreating an inflammatory disorder or a TNFα-related disorder in asubject in need thereof, said method comprising the step ofadministering the pharmaceutical composition of claim 17 to saidsubject.
 19. The method according to claim 18, wherein said inflammatorydisorder is an inflammatory disorder of the gastrointestinal tract. 20.The method according to claim 19, wherein said inflammatory disorder ofthe gastrointestinal tract is inflammatory bowel disease.
 21. The methodaccording to claim 19, wherein said inflammatory disorder of thegastrointestinal tract is Crohn's disease or ulcerative colitis.
 22. Theantibody or functional fragment of claim 1, wherein the sum of (i) thenumber of amino acids in framework regions I, II, and III of thevariable light domain of said antibody or functional fragment that aredifferent from the respective human Vκ1 consensus sequences SEQ ID NOs:56, 57, and 58, and (ii) the number of amino acids in framework regionIV of the variable light domain of said antibody or functional fragmentthat are different from the most similar human λ germline-based sequenceselected from SEQ ID NOs: 59, 60, 61, and 62, is less than 4.